Remote loading of sparingly water-soluble drugs into liposomes

ABSTRACT

The present invention provides liposome compositions containing sparingly soluble drugs that are used to treat life-threatening diseases. A preferred method of encapsulating a drug inside a liposome is by remote or active loading. Remote loading of a drug into liposomes containing a transmembrane electrochemical gradient is initiated by co-mixing a liposome suspension with a solution of drug, whereby the neutral form of the compound freely enters the liposome and becomes electrostatically charged thereby preventing the reverse transfer out of the liposome. There is a continuous build-up of compound within the liposome interior until the electrochemical gradient is dissipated or all the drug is encapsulated in the liposome. However, this process as described in the literature has been limited to drugs that are freely soluble in aqueous solution or solubilized as a water-soluble complex. This invention describes compositions and methods for remote loading drugs with low water solubility (&lt;2 mg/mL). In the preferred embodiment the drug in the solubilizing agent is mixed with the liposomes in aqueous suspension so that the concentration of solubilizing agent is lowered to below its capacity to completely solubilize the drug. This results in the drug precipitating but remote loading capability is retained. The process is scalable and, in liposomes in which the lipid composition and remote loading agent are optimized, the resulting drug-loaded liposomes are characterized by a high drug-to-lipid ratios and prolonged drug retention when the liposome encapsulated drug is administered to a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Patent Application No. 61/759,914 filed Feb. 1, 2013, whichis incorporated herein by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

This invention relates to the fields of pharmaceutical compositions,methods for making them and the uses of the resulting compositions indrug therapy. The pharmaceutical compositions include the activetherapeutic agent encapsulated within the aqueous interior of a liposomevesicle.

DESCRIPTION OF THE RELATED ART

The pharmaceutical industry, in its quest for improved drugs, hasgenerated a large number of potent compounds that are sparingly solublein water, the ubiquitous solvent that makes life possible. The low watersolubility of these new drugs has made it difficult to deliver them inanimals including humans. This has created the need for drug deliverysystems that can solubilize sparingly water-soluble drugs to enable totheir delivery in the body.

Liposomes are vesicle structures usually composed of a bilayer membraneof amphipathic molecules such as, phospholipids, entrapping an aqueouscore. The diameters and morphology of various types of liposomes areillustrated in FIG. 1. Drugs are either encapsulated in the aqueous coreor interdigitated in the bilayer membrane. Drugs interdigitated in themembrane transfer out of the liposome when it is diluted into the body.Importantly, drugs that are encapsulated in the aqueous core or held incomplexes in the aqueous core are retained substantially longer thandrugs in the bilayer. The use of liposomes with drugs encapsulated inthe aqueous core for drug delivery is well established (D. Drummond etal., J. Pharm. Sci., (2008) 97(11):4696-4740, PMID 10581328).

A variety of loading methods for encapsulating functional compounds,particularly drugs, in liposomes is available. Hydrophilic compounds forexample can be encapsulated in liposomes by hydrating a mixture of thefunctional compounds and vesicle-forming lipids. This technique iscalled passive loading. The functional compound is encapsulated in theliposome as the nanoparticle is formed. The available lipid vesicle(liposome) production procedures are satisfactory for most applicationswhere water-soluble drugs are encapsulated (G. Gregoriadis, Ed.,Liposome Technology, (2006) Liposome Preparation and Related Techniques,3rd Ed.) However, the manufacture of lipid vesicles that encapsulatedrugs sparing water-soluble (e.g., with a water solubility less than 2mg/mL) in the aqueous inner compartment of the liposome is exceedinglydifficult (D. Zucker et al., Journal of Controlled Release (2009)139:73-80, PMID 19508880).

Passive loading of lipophilic and to a lesser extent amphiphilicfunctional compounds is somewhat more efficient than hydrophilicfunctional compounds because they partition in both the lipid bilayerand the intraliposomal (internal) aqueous medium. However, using passiveloading, the final functional-compound-to-lipid ratio as well as theencapsulation efficiency are generally low. The concentration of drug inthe liposome equals that of the surrounding fluid and drug not entrappedin the internal aqueous medium is washed away after encapsulation.Moreover drugs loaded into the bilayer are released from the liposomevery rapidly when the liposome is injected into a subject. For sustainedrelease of the drug in a patient it is preferable that the drug isencapsulated within the interior of the lipsosome.

Certain hydrophilic or amphiphilic compounds can be loaded intopreformed liposomes using transmembrane pH- or ion-gradients (D. Zuckeret al., Journal of Controlled Release (2009) 139:73-80). This techniqueis called active or remote loading. Compounds amenable to active loadingshould be able to change from an uncharged form, which can diffuseacross the liposomal membrane, to a charged form that is not capablethereof. Typically, the functional compound is loaded by adding it to asuspension of liposomes prepared to have a lower inside/higher outsidepH- or ion-gradient. Via active loading, a highfunctional-compound-to-lipid mass ratio and a high loading efficiency(up to 100%) can be achieved. Examples are active loading of anticancerdrugs doxorubicin, daunorubicin, and vincristine (P. R. Cullis et al.,Biochimica et Biophysica Acta, (1997) 1331:187-211, and referencestherein).

Hydrophobic drugs are only considered capable of loading into liposomesthrough membrane intercalation via some passive loading/assemblymechanism. Wasan et al. states “Agents that have hydrophobic attributescan intercalate into the lipid bilayer and this can be achieved byadding the agent to the preformed liposomes.” in a description of theuse of micelles to transfer sparingly soluble agents to a liposomebilayer (US 2009/0028931).

Remote loading of a sparingly soluble drug into a liposome underconditions where the drug is above its solubility limit and is in theform of a precipitate is an unexpected event. D. Zucker et al., Journalof Controlled Release (2009) 139:73-80 states “Hydrophobic molecules mayaggregate, and these aggregates have low permeability across theliposomal membrane. Thus, when the non-polar/polar surface area ratiois >2.31 (see FIG. 4 in Zucker et al. Journal of Controlled Release(2009) 139:73-80), it is necessary that the drug would have a reasonablesolubility, >1.9 mM, in order to achieve high loading because onlysoluble uncharged molecules can enter the liposome.” (D. Zucker et al.,Journal of Controlled Release (2009) 139:73-80).

To date, a method has not been developed for the active loading of theaqueous core of a liposome with a sparingly water-soluble agent from aprecipitate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the diameters and morphology of various types ofliposomes.

FIG. 2. Liposome formulations composed of HSPC/Chol/Peg-DSPE containingeither sodium sulfate (light shade) or ammonium sulfate (dark shade)were incubated with carfilzomib at two input drug-to-lipid ratios usingconditions described below. The liposomes were purified fromunencapsulated drug and the amount of encapsulated carfilzomib withinthe liposomes is shown, expressed as μg of carfilzomib per μmol lipid.

FIG. 3 is a bar graph showing a trapping agent effect on liposomeloading of carfilzomib.

FIG. 4 is a bar graph showing a method of drug introduction effect onliposome loading of carfilzomib.

FIG. 5 is a line graph showing carfilzomib loading from precipitatedemonstrated by reduction of light scattering at 600 nm.

FIG. 6 is a HPLC Chromatogram of Carfilzomib before loading intoliposomes (upper) and after loading into liposomes from a precipitateand then being released from liposomes using a reverse ammonium sulfategradient where it is converted back to a precipitate in theextraliposomal solution (lower).

FIG. 7 is a line graph showing liposome encapsulation efficiency as afunction of [DMSO]. The input drug-to-lipid ratio was 200 μg/μmol.

FIG. 8 is a line graph showing light scattering of carfilzomib solutionsa function of DMSO concentration. The concentration of carfilzomib was0.2 mg/mL.

FIG. 9 is a bar graph showing the effect of delay time between theformation of drug precipitate and liposome loading of the precipitate.

FIG. 10 is a line graph showing the effect of ammonium sulfate trappingagent concentration on liposome drug payload of carfilzomib loaded froma precipitate.

FIG. 11 is a line graph showing effect of ammonium sulfate trappingagent concentration on liposome loading efficiency of carfilzomib fromprecipitate.

FIG. 12 is a line graph loading insoluble carfilzomib precipitate intoliposomes using a triethylammonium sulfate gradient.

FIG. 13 is a line graph showing the transfer of insoluble carfilzomibprecipitate into liposomes by remote loading.

FIG. 14 is a bar graph showing the comparison of liposome loading ofaripiprazole when mixed with liposomes as a SBCD complex (Abilify) orwhen diluted from a stock DMSO solution directly into liposomes,creating a drug suspension.

FIG. 15 is a bar graph showing absorbance at 600 nm (scattering) of drugsolutions (dark bars) and liposome drug mixtures (gray bars). Therectangle indicates the samples where a substantial decrease inscattering was measured upon incubation with liposomes indicating drugloading.

FIG. 16 is a bar graph showing the loading efficiency of DFX in calciumacetate liposomes.

FIG. 17 is a plot showing DFX loading capacity in liposomes containingcalcium acetate as a trapping agent.

FIG. 18 is a plot showing DFX loading capacity in liposomes containingdifferent acetate trapping agents.

SUMMARY OF THE INVENTION

In utilizing liposomes for delivery of functional compounds, it isgenerally desirable to load the liposomes to high concentration,resulting in a high functional-compound-lipid mass ratio, since thisreduces the amount of liposomes to be administered per treatment toattain the required therapeutic effect, all the more since severallipids used in liposomes have a dose-limiting toxicity by themselves.The loading percentage is also of importance for cost efficiency, sincepoor loading results in a great loss of the active compound.

In an exemplary embodiment, the invention provides a liposome comprisinga liposomal lipid membrane encapsulating an internal aqueous medium. Theinternal aqueous medium comprises an aqueous solution of a complexbetween a trapping agent and a sparingly water-soluble therapeuticagent.

In a further exemplary embodiment, the invention provides pharmaceuticalformulations comprising a liposome of the invention. The formulationsinclude the liposome and a pharmaceutically acceptable diluent orexcipient. In various embodiments, the pharmaceutical formulation is ina unit dosage format, providing a unit dosage of the therapeutic agentencapsulated in the liposome.

In another exemplary embodiment, the invention provides methods ofmaking the liposomes of the invention. In various embodiments, there isprovided a method of remotely loading a liposome with an agent that issparingly water-soluble. The method comprises: a) incubating an aqueousmixture comprising: (i) a liposome suspension having a proton and/or iongradient that exists across the liposomal membrane; (ii) with an aqueoussuspension of a sparingly soluble drug (iii) wherein the drug suspensionis made by completely dissolving the drug in an aprotic solvent orpolyol and diluting it into the aqueous solution beyond the point ofdrug solubility where a precipitate is formed, wherein incubating thecombined liposome drug precipitate mixture for a period of time resultsin the drug accumulating within the liposome interior in response to theproton/ion gradient. The mixture used to load the liposome with theagent is prepared such that a proton- and/or ion-gradient exists acrossthe liposomal membrane between the internal aqueous membrane and theexternal aqueous medium. The incubating can be for any useful period butis preferably for a period of time sufficient to cause at least part ofthe insoluble drug precipitate to accumulate in the internal aqueousmedium under the influence of the proton and/or ion gradient.

Other embodiments, objects and advantages are set forth in the DetailedDescription that follows.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Introduction

In utilizing liposomes for delivery of functional compounds, it isgenerally desirable to load the liposomes to high concentration,resulting in a high agent-lipid mass ratio, since this reduces theamount of liposomes to be administered per treatment to attain therequired therapeutic effect of the agent, all the more since severallipids used in liposomes have a dose-limiting toxicity by themselves.The loading percentage is also of importance for cost efficiency, sincepoor loading results in an increase loss of agent during the loading ofthe agent into the liposome.

The present invention provides liposomes encapsulating agents, e.g.,sparingly water-soluble, methods of making such liposomes, formulationscontaining such liposomes and methods of making the liposomes andformulations of the invention.

In an exemplary embodiment, the invention provides a liposome having amembrane encapsulating an aqueous compartment. The liposome is preparedsuch that a proton- and/or ion-gradient exists across the liposomalmembrane between the internal aqueous compartment and the externalaqueous medium. The agent is dissolved in an aprotic solvent at aconcentration that when diluted in the liposome suspension to form theremote loading mixture its solubility in the suspension is exceeded andthe agent forms a precipitate. A portion of the agent precipitate isloaded into the liposome aqueous compartment using a proton- and/orion-gradient across the liposomal membrane between the internal aqueouscompartment and the external aqueous medium.

In some embodiments, essentially the entire amount of the insolubleagent precipitate in the remote loading mixture is loaded into theaqueous compartment of the liposome. In an exemplary embodiment, atleast about 95%, at least about 90%, at least about 85%, at least about80% or at least about 70% of the insoluble drug precipitate in theremote loading mixture is loaded into the aqueous compartment of theliposome.

Liposomes

The term liposome is used herein in accordance with its usual meaning,referring to microscopic lipid vesicles composed of a bilayer ofphospholipids or any similar amphipathic lipids encapsulating aninternal aqueous medium. The liposomes of the present invention can beunilamellar vesicles such as small unilamellar vesicles (SUVs) and largeunilamellar vesicles (LUVs), and multilamellar vesicles (MLV), typicallyvarying in size from 30 nm to 200 nm. No particular limitation isimposed on the liposomal membrane structure in the present invention.The term liposomal membrane refers to the bilayer of phospholipidsseparating the internal aqueous medium from the external aqueous medium.

Exemplary liposomal membranes useful in the current invention may beformed from a variety of vesicle-forming lipids, typically includingdialiphatic chain lipids, such as phospholipids, diglycerides,dialiphatic glycolipids, single lipids such as sphingomyelin andglycosphingolipid, cholesterol and derivates thereof, and combinationsthereof. As defined herein, phospholipids are amphiphilic agents havinghydrophobic groups formed of long-chain alkyl chains, and a hydrophilicgroup containing a phosphate moiety. The group of phospholipids includesphosphatidic acid, phosphatidyl glycerols, phosphatidylcholines,phosphatidylethanolamines, phosphatidylinositols, phosphatidylserines,and mixtures thereof. Preferably, the phospholipids are chosen from1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),dimyristoyl-phosphatidylcholine (DMPC), hydrogenated soyphosphatidylcholine (HSPC), soy phosphatidylcholine (SPC),dimyristoylphosphatidylglycerol (DMPG), disrearoylphosphatidylglycerol(DSPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) distearoylphosphatidylcholine (DSPC), egg yolk phosphatidylcholine (EYPC) orhydrogenated egg yolk phosphatidylcholine (HEPC), sterol modified lipids(SML), cationic lipids and inverse-zwitterlipids.

Liposomal membranes according to the present invention may furthercomprise ionophores like nigericin and A23187.

In the method according to the present invention, an exemplary liposomalphase transition temperature is between −25° C. and 100° C., e.g.,between 4° C. and 65° C. The phase transition temperature is thetemperature required to induce a change in the physical state of thelipids constituting the liposome, from the ordered gel phase, where thehydrocarbon chains are fully extended and closely packed, to thedisordered liquid crystalline phase, where the hydrocarbon chains arerandomly oriented and fluid. Above the phase transition temperature ofthe liposome, the permeability of the liposomal membrane increases.Choosing a high transition temperature, where the liposome would alwaysbe in the gel state, could provide a non-leaking liposomal composition,i.e. the concentration of the sparingly water-soluble agent in theinternal aqueous medium is maintained during exposure to theenvironment. Alternatively, a liposome with a transition temperaturebetween the starting and ending temperature of the environment it isexposed to provides a means to release the sparingly water-soluble agentwhen the liposome passes through its transition temperature. Thus, theprocess temperature for the active-loading technique typically is abovethe liposomal phase transition temperature to facilitate theactive-loading process. As is generally known in the art, phasetransition temperatures of liposomes can, among other parameters, beinfluenced by the choice of phospholipids and by the addition ofsteroids like cholesterol, lanosterol, cholestanol, stigmasterol,ergosterol, and the like. Hence, in an embodiment of the invention, amethod according to any of the foregoing is provided in which theliposomes comprise one or more components selected from differentphospholipids and cholesterol in several molar ratios in order to modifythe transition, the required process temperature and the liposomestability in plasma. Less cholesterol in the mixture will result in lessstable liposomes in plasma. An exemplary phospholipid composition of usein the invention comprises between about 10 and about 50 mol % ofsteroids, preferably cholesterol.

In accordance with the invention, liposomes can be prepared by any ofthe techniques now known or subsequently developed for preparingliposomes. For example, the liposomes can be formed by the conventionaltechnique for preparing multilamellar lipid vesicles (MLVs), that is, bydepositing one or more selected lipids on the inside walls of a suitablevessel by dissolving the lipids in chloroform and then evaporating thechloroform, and by then adding the aqueous solution which is to beencapsulated to the vessel, allowing the aqueous solution to hydrate thelipid, and swirling or vortexing the resulting lipid suspension. Thisprocess engenders a mixture including the desired liposomes.Alternatively, techniques used for producing large unilamellar lipidvesicles (LUVs), such as reverse-phase evaporation, infusion procedures,and detergent dilution, can be used to produce the liposomes. A reviewof these and other methods for producing lipid vesicles can be found inthe text Liposome Technology, Volume I, Gregory Gregoriadis Ed., CRCPress, Boca Raton, Fla., (1984), which is incorporated herein byreference. For example, the lipid-containing particles can be in theform of steroidal lipid vesicles, stable plurilamellar lipid vesicles(SPLVs), monophasic vesicles (MPVs), or lipid matrix carriers (LMCs). Inthe case of MLVs, if desired, the liposomes can be subjected to multiple(five or more) freeze-thaw cycles to enhance their trapped volumes andtrapping efficiencies and to provide a more uniform interlamellardistribution of solute.

Following liposome preparation, the liposomes are optionally sized toachieve a desired size range and relatively narrow distribution ofliposome sizes. A size range of about 20-200 nanometers allows theliposome suspension to be sterilized by filtration through aconventional filter, typically a 0.22 or 0.4 micron filter. The filtersterilization method can be carried out on a high through-put basis ifthe liposomes have been sized down to about 20-200 nanometers. Severaltechniques are available for sizing liposomes to a desired size.Sonicating a liposome suspension either by bath or probe sonicationproduces a progressive size reduction down to small unilamellar vesiclesless than about 50 nanometer in size. Homogenization is another methodwhich relies on shearing energy to fragment large liposomes into smallerones. In a typical homogenization procedure, multilamellar vesicles arerecirculated through a standard emulsion homogenizer until selectedliposome sizes, typically between about 50 and 500 nanometers, areobserved. In both methods, the particle size distribution can bemonitored by conventional laser-beam particle size determination.Extrusion of liposome through a small-pore polycarbonate membrane or anasymmetric ceramic membrane is also an effective method for reducingliposome sizes to a relatively well-defined size distribution.Typically, the suspension is cycled through the membrane one or moretimes until the desired liposome size distribution is achieved. Theliposomes may be extruded through successively smaller-pore membranes,to achieve a gradual reduction in liposome size. Alternativelycontrolled size liposomes can be prepared using microfluidic techniqueswherein the lipid in an organic solvent such as ethanol orethanol-aprotic solvent mixtures is rapidly mixed with the aqueousmedium, so that the organic solvent/water ratio is less than 30%, in amicrochannel with dimensions less than 300 microns and preferable lessthan 150 microns in wide and 50 microns in height. The organic solventis then removed from the liposomes by dialysis. Other useful sizingmethods such as sonication, solvent vaporization or reverse phaseevaporation are known to those of skill in the art.

Exemplary liposomes for use in various embodiments of the invention havea size of from about 30 nanometers to about 40 microns.

The internal aqueous medium, as referred to herein, typically is theoriginal medium in which the liposomes were prepared and which initiallybecomes encapsulated upon formation of the liposome. In accordance withthe present invention, freshly prepared liposomes encapsulating theoriginal aqueous medium can be used directly for active loading.Embodiments are also envisaged however wherein the liposomes, afterpreparation, are dehydrated, e.g. for storage. In such embodiments thepresent process may involve addition of the dehydrated liposomesdirectly to the external aqueous medium used to create the transmembranegradients. However it is also possible to hydrate the liposomes inanother external medium first, as will be understood by those skilled inthe art. Liposomes are optionally dehydrated under reduced pressureusing standard freeze-drying equipment or equivalent apparatus. Invarious embodiments, the liposomes and their surrounding medium arefrozen in liquid nitrogen before being dehydrated and placed underreduced pressure. To ensure that the liposomes will survive thedehydration process without losing a substantial portion of theirinternal contents, one or more protective sugars are typically employedto interact with the lipid vesicle membranes and keep them intact as thewater in the system is removed. A variety of sugars can be used,including such sugars as trehalose, maltose, sucrose, glucose, lactose,and dextran. In general, disaccharide sugars have been found to workbetter than monosaccharide sugars, with the disaccharide sugarstrehalose and sucrose being most effective. Other more complicatedsugars can also be used. For example, aminoglycosides, includingstreptomycin and dihydrostreptomycin, have been found to protectliposomes during dehydration. Typically, one or more sugars are includedas part of either the internal or external media of the lipid vesicles.Most preferably, the sugars are included in both the internal andexternal media so that they can interact with both the inside andoutside surfaces of the liposomes' membranes. Inclusion in the internalmedium is accomplished by adding the sugar or sugars to the buffer whichbecomes encapsulated in the lipid vesicles during the liposome formationprocess. In these embodiments the external medium used during the activeloading process should also preferably include one or more of theprotective sugars

As is generally known to those skilled in the art, polyethylene glycol(PEG)-lipid conjugates have been used extensively to improve circulationtimes for liposome-encapsulated functional compounds, to avoid or reducepremature leakage of the functional compound from the liposomalcomposition and to avoid detection of liposomes by the body's immunesystem. Attachment of PEG-derived lipids onto liposomes is calledPEGylation. Hence, in an exemplary embodiment of the invention, theliposomes are PEGylated liposomes. PEGylation can be accomplished byincubating a reactive derivative of PEG with the target liposomes.Suitable PEG-derived lipids according to the invention, includeconjugates of DSPE-PEG, functionalized with one of carboxylic acids,glutathione (GSH), maleimides (MAL), 3-(2-pyridyldithio) propionic acid(PDP), cyanur, azides, amines, biotin or folate, in which the molecularweight of PEG is between 2000 and 5000 g/mol. Other suitable PEG-derivedlipids are mPEGs conjugated with ceramide, having either C₈- or C₁₆-tails, in which the molecular weight of mPEG is between 750 and 5000daltons. Still other appropriate ligands are mPEGs or functionalizedPEGs conjugated with glycerophospholipds like1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and the like.PEGylation of liposomes is a technique generally known by those skilledin the art.

In various embodiments, the liposomes are PEGylated with DSPE-PEG-GSHconjugates (up to 5 mol %) and/or DSPE-mPEG conjugates (wherein themolecular weight of PEG is typically within the range of 750-5000daltons, e.g. 2000 daltons). The phospholipid composition of anexemplary PEGylated lipsome of the invention may comprise up to 5-20 mol% of PEG-lipid conjugates.

Furthermore, in certain embodiments, one or more moieties thatspecifically target the liposome to a particular cell type, tissue orthe like are incorporated into the membrane. Targeting of liposomesusing a variety of targeting moieties (e.g., ligands, receptors andmonoclonal antibodies) has been previously described. Suitable examplesof such targeting moieties include hyaluronic acid, anti-ErbB familyantibodies and antibody fragments, lipoprotein lipase (LPL),[α]2-macroglobulin ([a]2M), receptor associated protein (RAP),lactoferrin, desmoteplase, tissue- and urokinase-type plasminogenactivator (tPA/uPA), plasminogen activator inhibitor (PAI-I),tPA/uPA:PAI-1 complexes, melanotransferrin (or P97), thrombospondin 1and 2, hepatic lipase, factor Vila/tissue-factor pathway inhibitor(TFPI), factor Villa, factor IXa, A[β]1-40, amyloid-[β] precursorprotein (APP), CI inhibitor, complement C3, apolipoproteinE (apoE),pseudomonas exotoxin A, CRM66, HIV-I Tat protein, rhinovirus, matrixmetalloproteinase 9 (MMP-9), MMP-13 (collagenase-3), spingolipidactivator protein (SAP), pregnancy zone protein, antithrombin III,heparin cofactor II, [α]1-antitrypsin, heat shock protein 96 (HSP-96),platelet-derived growth factor (PDGF), apolipoproteinJ (apoJ, orclusterin), A[β] bound to apoJ and apoE, aprotinin, angiopep-2(TFFYGGSRGKRNNFKTEEY), very-low-density lipoprotein (VLDL), transferrin,insulin, leptin, an insulin-like growth factor, epidermal growthfactors, lectins, peptidomimetic and/or humanized monoclonal antibodies,dingle chain antibodies or peptides specific for said receptors (e.g.,sequences HAIYPRH and THRPPMWSPVWP that bind to the human transferrinreceptor, or anti-human transferrin receptor (TfR) monoclonal antibodyA24), hemoglobin, non-toxic portion of a diphtheria toxin polypeptidechain, all or a portion of the diphtheria toxin B chain, all or aportion of a non-toxic mutant of diphtheria toxin CRM197, apolipoproteinB, apolipoprotein E (e.g., after binding to polysorb-80 coating),vitamin D-binding protein, vitamin A/retinol-binding protein, vitaminB12/cobalamin plasma carrier protein, glutathione and transcobalamin-B12.

Targeting mechanisms generally require that the targeting agents bepositioned on the surface of the liposome in such a manner that thetarget moieties are available for interaction with the target, forexample, a cell surface receptor. In an exemplary embodiment, theliposome is manufactured to include a connector portion incorporatedinto the membrane at the time of forming the membrane. An exemplaryconnector portion has a lipophilic portion which is firmly embedded andanchored in the membrane. An exemplary connector portion also includes ahydrophilic portion which is chemically available on the aqueous surfaceof the liposome. The hydrophilic portion is selected so that it will bechemically suitable to form a stable chemical bond with the targetingagent, which is added later. Techniques for incorporating a targetingmoiety in the liposomal membrane are generally known in the art.

In an exemplary embodiment, the liposome includes HSPC, cholesterol,PEG-DSPE and a combination thereof. In an exemplary embodiment, theliposome includes from about 50 mol to about 70 mol HSPC, from about 30mol to about 50 mol cholesterol and from about 1 mol to about 10 molPEG-DSPE. In one embodiment, the liposome includes about 60 mol HSPC,about 40 mol cholesterol and about 5 mol PEG DSPE.

Sparingly Water-Soluble Agent

As indicated above, the present invention provides liposomesencapsulating a a sparingly water-soluble agent. In the context of thepresent invention the term ‘sparingly water-soluble’ means beinginsoluble or having a very limited solubility in water, more inparticular having an aqueous solubility of less than 2 mg/mL, e.g., lessthan 1.9 mg/mL, e.g., having an aqueous solubility of less than 1 mg/mL.As used herein, water solubilities refer to solubilities measured atambient temperature, which is typically about 20° C. In an exemplaryembodiment, the water solubility of the agent is measured at about pH=7.

According to an exemplary embodiment of the invention, the sparinglywater-soluble agent is a therapeutic agent selected from the group of atherapeutic is selected from a group consisting of an anthracyclinecompound, a camptothecin compound, a vinca alkaloid, an ellipticinecompound, a taxane compound, a wortmannin compound, a geldanamycincompound, a pyrazolopyrimidine compound, a peptide-based compound suchas carfilzomib, a steroid compound, a derivative of any of theforegoing, a pro-drug of any of the foregoing, and an analog of any ofthe foregoing.

Exemplary small molecule compounds having a water solubility less thanabout 2 mg/mL include, but are not limited to, carfilzomib,voriconazole, amiodarone, ziprasidone, aripiprazole, imatinib,lapatinib, cyclopamine, oprozomib, CUR-61414, PF-05212384, PF-4691502,toceranib, PF-477736, PF-337210, sunitinib, SU14813, axitinib, AG014699,veliparib, MK-4827, ABT-263, SU11274, PHA665752, Crizotinib, XL880,PF-04217903, XR5000, AG14361, veliparib, bosutunib, PD-0332991,PF-01367338, AG14361, NVP-ADW742, NVP-AUY922, NVP-LAQ824, NVP-TAE684,NVP-LBH589, erubulin, doxorubicin, daunorubicin, mitomycin C,epirubicin, pirarubicin, rubidomycin, carcinomycin, N-acetyladriamycin,rubidazone, 5-imido daunomycin, N-acetyl daunomycin, daunory line,mitoxanthrone, camptothecin, 9-aminocamptothecin, 7-ethylcamptothecin,7-Ethyl-10-hydroxy-camptothecin, 10-hydroxycamptothecin,9-nitrocamptothecin, 1O,11-methylenedioxycamptothecin,9-amino-1O,11-methylenedioxycamptothecin,9-chloro-10,11-methylenedioxycamptothecin, irinotecan, lurtotecan,silatecan,(7-(4-methylpiperazinomethylene)-10,II-ethylenedioxy-20(S)-camptothecin,7-(4-methylpiperazinomethylene)-10,II-methylenedioxy-20(S)-camptothecin,7-(2-N-isopropylamino)ethyl)-(20S)-camptothecin, CKD-602, vincristine,vinblastine, vinorelbine, vinflunine, vinpocetine, vindesine,ellipticine, 6-3-aminopropyl-ellipticine,2-diethylaminoethyl-ellipticinium, datelliptium, retelliptine,paclitaxel, docetaxel, diclofenac, bupivacaine,17-Dimethylaminoethylamino-17-demethoxygeldanamycin, cetirizine,fexofenadine, primidone and other catecholamines, epinephrine,(S)-2-(2,4-dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylicacid (deferitrin),(S)-4,5-dihydro-2-(3-hydroxy-2-pyridinyl)-4-methyl-4-thiazolecarboxylicacid (desferrithiocin),(S)-4,5-dihydro-2-[2-hydroxy-4-(3,6,9,12-tetraoxatridecyloxy)phenyl]-4-methyl-4-thiazolecarboxylicacid,(S)-4,5-dihydro-2-[2-hydroxy-4-(3,6-dioxaheptyloxy)phenyl]-4-methyl-4-thiazolecarboxylicacid, ethyl(S)-4,5-dihydro-2-[2-hydroxy-4-(3,6-dioxaheptyloxy)phenyl]-4-methyl-4-thiazolecarboxylate,(S)-4,5-dihydro-2-[2-hydroxy-3-(3,6,9-trioxadecyloxy)]-4-methyl-4-thiazolecarboxylicacid, desazadesferrithiocin salts, prodrugs and derivatives of thesemedicinal compounds and mixtures thereof.

An exemplary therapeutic agent is selected from: an antihistamineethylenediamine derivative, bromphenifamine, diphenhydramine, ananti-protozoal drug, quinolone, iodoquinol, an amidine compound,pentamidine, an antihelmintic compound, pyrantel, an anti-schistosomaldrug, oxaminiquine, an antifungal triazole derivative, fliconazole,itraconazole, ketoconazole, miconazole, an antimicrobial cephalosporin,chelating agents, deferoxamine, deferasirox, deferiprone, FBS0701,cefazolin, cefonicid, cefotaxime, ceftazimide, cefuoxime, anantimicrobial beta-lactam derivative, aztreopam, cefmetazole, cefoxitin,an antimicrobial of erythromycin group, erythromycin, azithromycin,clarithromycin, oleandomycin, a penicillin compound, benzylpenicillin,phenoxymethylpenicillin, cloxacillin, methicillin, nafcillin, oxacillin,carbenicillin, a tetracycline compound, novobiocin, spectinomycin,vancomycin; an antimycobacterial drug, aminosalicycic acid, capreomycin,ethambutol, isoniazid, pyrazinamide, rifabutin, rifampin, clofazimine,an antiviral adamantane compound, amantadine, rimantadine, a quinidinecompound, quinine, quinacrine, chloroquine, hydroxychloroquine,primaquine, amodiaquine, mefloquine, an antimicrobial, qionolone,ciprofloxacin, enoxacin, lomefloxacin, nalidixic acid, norfloxacin,ofloxacin, a sulfonamide; a urinary tract antimicrobial, nitrofurantoin,trimetoprim; anitroimidazoles derivative, metronidazole, a cholinergicquaternary ammonium compound, ambethinium, neostigmine, physostigmine,an anti-Alzheimer aminoacridine, tacrine, an anti-parkinsonal drug,benztropine, biperiden, procyclidine, trihexylhenidyl, ananti-muscarinic agent, atropine, hyoscyamine, scopolamine,propantheline, an adrenergic compound, dopamine, serotonin, a hedgehoginhibitor, albuterol, dobutamine, ephedrine, epinephrine,norepinephrine, isoproterenol, metaproperenol, salmetrol, terbutaline, aserotonin reuptake inhibitor, an ergotamine derivative, a myorelaxant, acurare series, a central action myorelaxant, baclophen, cyclobenzepine,dentrolene, nicotine, a nicotine receptor antagonist, abeta-adrenoblocker, acebutil, amiodarone, abenzodiazepine compound,ditiazem, an antiarrhythmic drug, diisopyramide, encaidine, a localanesthetic compound, procaine, procainamide, lidocaine, flecaimide,quinidine, an ACE inhibitor, captopril, enelaprilat, Hsp90 inhibitor,fosinoprol, quinapril, ramipril; an opiate derivative, codeine,meperidine, methadone, morphine, an antilipidemic, fluvastatin,gemfibrosil, an HMG-coA inhibitor, pravastatin, a hypotensive drug,clonidine, guanabenz, prazocin, guanethidine, granadril, hydralazine, anon-coronary vasodilator, dipyridamole, an acetylcholine esteraseinhibitor, pilocarpine, an alkaloid, physostigmine, neostigmine, aderivative of any of the foregoing, a pro-drug of any of the foregoing,and analog of any of the foregoing.

This list of agents, however, is not intended to limit the scope of theinvention. In fact, the compound encapsulated within the liposome can beany sparingly water-soluble amphipathic weak base or amphipathic weakacid. As noted above, embodiments wherein the sparingly water-solubleagent is not a pharmaceutical or medicinal agent are also encompassed bythe present invention.

Typically, within the context of the present invention, sparinglywater-soluble amphipathic weak bases have an octanol-water distributioncoefficient (logD) at pH 7 between −2.5 and 2 and pKa<11, whilesparingly water-soluble amphipathic weak acids have a logD at pH 7between −2.5 and 2 and pKa>3.

Typically, the terms weak base and weak acid, as used in the foregoing,respectively refer to compounds that are only partially protonated ordeprotonated in water. Examples of protonable agents include compoundshaving an amino group, which can be protonated in acidic media, andcompounds which are zwitterionic in neutral media and which can also beprotonated in acidic environments. Examples of deprotonable agentsinclude compounds having a carboxy group, which can be deprotonated inalkaline media, and compounds which are zwitterionic in neutral mediaand which can also be deprotonated in alkaline environments.

The term zwitterionic refers to compounds that can simultaneously carrya positive and a negative electrical charge on different atoms. The termamphipathic, as used in the foregoing is typically employed to refer tocompounds having both lipophilic and hydrophilic moieties. The foregoingimplies that aqueous solutions of compounds being weak amphipathic acidsor bases simultaneously comprise charged and uncharged forms of saidcompounds. Only the uncharged forms may be able to cross the liposomalmembrane.

When agents of use in the present invention contain relatively basic oracidic functionalities, salts of such compounds are included in thescope of the invention. Salts can be obtained by contacting the neutralform of such compounds with a sufficient amount of the desired acid orbase, either neat or in a suitable inert solvent. Examples of salts forrelative acidic compounds of the invention include sodium, potassium,calcium, ammonium, organic amino, or magnesium salts, or a similarsalts. When compounds of the present invention contain relatively basicfunctionalities, acid addition salts can be obtained by contacting theneutral form of such compounds with a sufficient amount of the desiredacid, either neat or in a suitable inert solvent. Examples of acidaddition salts include those derived from inorganic acids likehydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from organic acids like acetic, propionic,isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric,lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric,tartaric, methanesulfonic, and the like. Also included are salts ofamino acids such as arginate and the like, and salts of organic acidslike glucuronic or galactunoric acids and the like (see, for example,Berge et al., Journal of Pharmaceutical Science 1977, 66: 1-19). Certainspecific compounds of the present invention contain both basic andacidic functionalities that allow the compounds to be converted intoeither base or acid addition salts.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents, but otherwise the salts are equivalentto the parent form of the compound for the purposes of the presentinvention.

An exemplary agent is a small organic molecule with a molecular weightbetween about 100 Da and 3000 Da.

In the embodiment in which a unit dosage format is formed, the liposomewill, in exemplary embodiments include from about 1 mg to about 500 mgof the approved agent, e.g, from about 1 mg to about 200 mg, e.g., fromabout 5 mg to about 100 mg, e.g., from about 10 mg to about 60 mg.

In an exemplary embodiment, the unit dosage includes the approved agentcarfilzomib and it is present in the liposome in an amount of from about40 mg to about 80 mg, e.g., from about 50 mg to about 70 mg. In anexemplary embodiment, the carfilzomib is present in about 60 mg.

Active Loading

The process of active loading, involves the use of transmembranepotentials. The principle of active loading, in general, has beendescribed extensively in the art. The terms active-loading andremote-loading are synonymous and will be used interchangeably.

During active loading, the precipitate of the sparingly water-solubleagent is transferred from the external aqueous medium across theliposomal membrane to the internal aqueous medium by a transmembraneproton- or ion-gradient. The term gradient of a particular compound asused herein refers to a discontinuous increase of the concentration ofsaid compound across the liposomal membrane from outside (externalaqueous medium) to inside the liposome (internal aqueous medium).

To create the concentration gradient, the liposomes are typically formedin a first liquid, typically aqueous, phase, followed by replacing ordiluting said first liquid phase. The diluted or new external medium hasa different concentration of the charged species or a totally differentcharged species, thereby establishing the ion- or proton-gradient.

The replacement of the external medium can be accomplished by varioustechniques, such as, by passing the lipid vesicle preparation through agel filtration column, e.g., a Sephadex or Sepharose column, which hasbeen equilibrated with the new medium, or by centrifugation, dialysis,or related techniques.

The efficiency of active-loading into liposomes depends, among otheraspects, on the chemical properties of the complex to be loaded and thetype and magnitude of the gradient applied. In an embodiment of theinvention, a method as defined in any of the foregoing is providedemploying a gradient across the liposomal membrane, in which thegradient is chosen from a pH-gradient, a sulfate-, phosphate-,phosphonate-, citrate-, or acetate-salt gradient, an EDTA-ion gradient,an ammonium-salt gradient, an alkylated, e.g methyl-, ethyl-, propyl-and amyl, ammonium-salt gradient, a Mn²⁺-, Cu²⁺-, Na⁺-, K⁺-gradient,with or without using ionophores, or a combination thereof. Theseloading techniques have been extensively described in the art.

Preferably, the internal aqueous medium of pre-formed, i.e., unloaded,liposomes comprises a so-called active-loading buffer which containswater and, dependent on the type of gradient employed during activeloading, may further comprise a sulfate-, phosphate-, phosphonate-,citrate-, or acetate-salt, an ammonium-salt, an alkylated, e.g.,methyl-, ethyl-, propyl- and amyl, ammonium-salt, an Fe⁺²-, Mn²⁺-, Cu²⁺-Na⁺ and/or K⁺-salt, an EDTA-ion salt, and optionally a pH-buffer tomaintain a pH-gradient. The salts may be polymeric such as dextransulfate, polyethyleneimine, polyamidoamine dendrimers, the 1.5carboxylate terminal version of polyamidoamines, polyphosphates, lowmolecular weight heparin, or hyaluronic acid. In an exemplaryembodiment, the concentration of salts in the internal aqueous medium ofunloaded liposomes is between 1 and 1000 mM.

The external aqueous medium, used to establish the transmembranegradient for active loading, comprises water, the precipitate of thesparingly water-soluble agent(s) to be loaded, and optionally sucrose,saline or some other agent to adjust the osmolarity and/or a chelatorlike EDTA to aid ionophore activity, more preferably sucrose and/orEDTA.

In an exemplary embodiment the gradient is chosen from an amine or ametal salt of a member selected from a carboxylate, sulfate,phosphonate, phosphate or an acetate. As is generally known by thoseskilled in the art, transmembrane pH- (lower inside, higher outside pH)or cation acetate-gradients can be used to actively load amphiphilicweak acids. Amphipathic weak bases can also be actively loaded intoliposomes using an ammonium sulfate- or triethylamine sulfate orammonium chloride-gradient.

Carboxylates of use in the invention include, without limitation,carboxylate, citrate, diethylenetriaminepentaaceetate, melletic acetate,1,2,3,4-butanetetracarboxylate, benzoate, isophalate, phthalate,3,4-bis(carboxymethyl)cyclopentanecarboxylate, the carboxylategeneration of polyamidoamine dendrimers, benzenetricarboxylates,benzenetetracarboxylates, ascorbate, glucuronate, and ulosonate.

Sulfates of use in the invention include, but are not limited to,sulfate, 1,5-naphthalenedisulfonate, dextran sulfate, sulfobutlyetherbeta cyclodextrin, sucrose octasulfate benzene sulfonate,poly(4-styrenesulfonate) trans resveratrol-trisulfate.

Phosphates and phosphonates of use in the invention include, but are notlimited to, phosphate, hexametaphosphate, phosphate glasses,polyphosphates, triphosphate, trimetaphosphate, bisphosphonates,ethanehydroxy bisphosphonate, inositol hexaphosphate

Exemplary salts of use in the invention include a mixture ofcarboxylate, sulfate or phosphate including but not limited to:2-carboxybenensulfonate, creatine phosphate, phosphocholine, carnitinephosphate, the carboxyl generation of polyamidoamines.

Amines of use in the invention include, but are not limited to,monoamines, polyamines, trimethylammonium, triethylammonium, tributylammonium, diethylmethylammonium, diisopropylethyl ammonium,triisopropylammonium, N-methylmorpholinium, N-ethylmorpholinium,N-hydroxyethylpiperidinium, N-methylpyrrolidinium,N,N-dimethylpiperazinium, isopropylethylammonium,isopropylmethylammonium, diisopropylammonium, tert-butylethylammonium,dicychohexylammonium, protonized forms of morpholine, pyridine,piperidine, pyrrolidine, piperazine, imidazole, tert-butylamine,2-amino-2-methylpropanol, 2-amino-2-methyl-propandiol,tris-(hydroxyethyl)-aminomethane, diethyl-(2-hydroxyethyl)amine,tris-(hydroxymethyl)-aminomethane tetramethylammonium,tetraethylammonium, N-methylglucamine and tetrabutylammonium,polyethyleneimine, and polyamidoamine dendrimers.

Depending upon the permeability of the lipid vesicle membranes, the fulltransmembrane potential corresponding to the concentration gradient willeither form spontaneously or a permeability enhancing agent, e.g., aproton ionophore can be added to the medium. If desired, thepermeability enhancing agent can be removed from the liposomepreparation after loading with the complex is complete usingchromatography or other techniques.

Typically the temperature of the medium during active loading is betweenabout −25° C. and about 100° C., e.g., between about 0° C. and about 70°C., e.g., between about 4° C. and 65° C.

The encapsulation or loading efficiency, defined as encapsulated amount(e.g., as measured in grams of agent/moles of phospholipid or g ofdrug/g total lipid) of the sparingly water-soluble agent in the internalaqueous phase divided by the initial amount in the external aqueousphase multiplied by 100%, is at least 10%, preferably at least 50%, atleast 90%.

In an exemplary embodiment, the invention provides a method of loading asparingly water-soluble agent into a liposome. An exemplary methodcomprises, contacting an aqueous suspension of said liposome with anaqueous suspension of the agent under conditions appropriate toencapsulate the sparingly water-soluble agent in said liposome. Theliposome has an internal aqueous environment encapsulated by a lipidmembrane. The aqueous suspension of the liposome comprises a gradientselected from a proton gradient, an ion gradient and a combinationthereof across the membrane. The sparingly water-soluble agent and theliposome suspension are incubated under conditions and for a selectedtime period appropriate for the sparingly water-soluble agent totraverse the membrane and concentrate in the internal aqueousenvironment, thereby forming said pharmaceutical formulation.

In various embodiments, the reaction mixture above is incubated for aselected period of time and the pH gradient, sulfate gradient, phosphategradient, phosphonate gradient, carboxylate gradient (citrate gradient,acetate gradient, etc.), EDTA ion gradient, ammonium salt gradient,alkylated ammonium salt gradient, Mg⁺², Mn⁺², Cu⁺², Na⁺, K⁺ gradient ora combination thereof, exists across the liposomal membrane during theincubating.

In exemplary embodiments of the invention, the sparingly water-solubletherapeutic agent is not covalently attached to a component of theliposome, nor is it covalently attached to any component of the pH orsalt gradient used to form the liposomal agent preparation of theinvention.

Aprotic Solvent

In an exemplary embodiment, the sparingly water-soluble agent iscompletely dissolved in an aprotic solvent that is miscible with water.The agent solution is added to the aqueous liposome suspension at aconcentration that is greater than the solubility of the drug agent ineither the liposome suspension or the liposome suspension/aproticsolvent mixture, thus a precipitate is formed. Exemplary aproticsolvents include dimethylsulfoxide, dioxane, tetrahydrofuran,dimethylformamide, acetonitrile, dimethylacetamide, sulfolane, gammabutyrolactone, pyrrolidones, 1-methyl-2-pyrrolidinone, methylpyrroline,ethylene glycol monomethyl ether, diethylene glycol monomethyl ether,PEG400 and polyethylene glycols.

Sparingly Water-Soluble Agent Precipitate

The invention provides methods for loading of an insoluble precipitateinside the aqueous internal compartment of the lipid membrane of aliposome. An exemplary precipitate is conceptualized as some insolubleportion of the agent in suspension. The insoluble portion is defined asa portion of the agent that is not solvated as indicated by any of thefollowing: any appearance of cloudiness greater than that of theliposome suspension in the absence of the agent, any degree of increasedlight scattering at a wavelength where the contents do not absorb light,such at 600 nm greater than the liposome suspension alone, any portionof the drug than can be isolated (pelleted) through centrifugation at arate below 12,000 RPM for 15 min, any portion of the drug agent than canbe isolated by filtration through 0.2 um filter.

Exemplary Formulation

In an exemplary embodiment, the invention provides a formulation of asparingly water-soluble agent encapsulated within the aqueous core of aliposome comprising cholesterol, PEG-DSPE and a lipid component with aphopshocholine headgroup and one or two fatty acid residues. In variousembodiments, the liposome comprises a combination of HSPC, cholesterol,and PEG-DSPE. In an exemplary embodiment, the liposome includes a molarratio of components of from about 50 mole percent to about 70 molepercent HSPC, from about 25 mole percent to about 50 mole percentcholesterol and from about 0 mole percent to about 10 mole percentPEG-DSPE. In one embodiment, the liposome includes a molar ratio ofcomponents about 60 mole percent HSPC, about 40 mole percent cholesteroland about 2.8 mole percent PEG-DSPE. In an exemplary embodiment, theagent is encapsulated in the liposome by active loading.

In a further exemplary embodiment, the invention provides a formulationof a sparingly water-soluble agent encapsulated within the aqueous coreof a liposome comprising a combination of sphingomyelin (SM),cholesterol, and PEG-DSPE. In an exemplary embodiment, the liposomeincludes a molar ratio of components from about 45 to about 75 SM, fromabout 25 mole percent to about 50 mole percent cholesterol and fromabout 0 mole percent to about 10 mole percent PEG-DSPE. In oneembodiment, the liposome includes a molar ratio of components of about55 mole percent SM, about 45 mole percent cholesterol and about 2.8 molepercent PEG-DSPE.

In an exemplary embodiment, the agent is carfilzomib or a salt, ananalog or derivative thereof. In an exemplary embodiment, thecarfilzomib:lipid ratio is from about 30 mg to about 90 mg ofcarfilzomib to from about 90 mg to about 250 mg of lipid. In anexemplary embodiment, the formulation the carfilzomid:lipid ratio isabout 60 mg carfilzomib to about 170 mg lipid.

In various embodiments, the carfilzomib (μg): lipid (μmol) ratio is fromabout 250 to about 450. In an exemplary embodiment, this ratio is fromabout 300 to about 400.

In an exemplary embodiment, the remote loading agent is an ammonium saltof a carbohydrate sulfate. In various embodiments, the remote loadingagent is triethylammonium dextran sulfate.

In an exemplary embodiment, the invention provides a pharmaceuticalformulation comprising about 60 mg of carfilzomib encapsulated within apopulation of liposomes whose mass is from about 90 mg to about 200 mg.The liposomes comprise a lipid membrane defining an internal aqueouscompartment. The carfilzomib is encapsulated within the aqueouscompartment defined by the lipid membrane. The lipid membrane has amolar ratio of components: (a) of about 55 mole percent sphingomyelin(SM); (b) about 45 mole percent cholesterol; and (c) about 2.8 molepercent PEG-(1,2-distearoyl-sn-glycero-3-phosphoethanolamine)(PEG-DSPE). The formulation is selected from a lyophilized formulationand a formulation in which said liposomes are suspended in apharmaceutically acceptable diluent.

In an exemplary embodiment, the invention provides a liposomeformulation of carfilzomib in which the carfilzomib has an in vivoT_(1/2) of from about 2 hours to about 12 hours, e.g., from about 3hours to about 6 hours, in a subject to whom it is administered. In anexemplary formulation, the carfilzomib is in either free or encapsulatedform.

An exemplary pharmaceutical formulation of the invention includes aliposome further encapsulating the carbohydrate sulfate. In an exemplaryembodiment, the carbohydrate sulfate is dextran sulfate.

In an exemplary embodiment, the formulation of the invention is formedby a method comprising: (a) preparing a suspension of the liposome in anaqueous solution of an ammonium salt of a carbohydrate sulfate, forminga first population of said ammonium salt encapsulated within saidliposome and a second population of said salt external to said liposome;(b) replacing the salt external to said liposome with an aqueous buffer,thereby forming a gradient of said carbohydrate sulfate across saidlipid membrane; and (c) adding a solution of carfilzomib in an aproticsolvent to the suspension formed in (b), forming a carfilzomibprecipitate capable of traversing said lipid membrane, concentrating insaid internal aqueous compartment, thereby encapsulating saidcarfilzomib.

In an exemplary embodiment, the ammonium salt of the carbohydratesulfate is triethylammonium dextran sulfate or ammonium dextran sulfate.

In an exemplary embodiment, the carfilzomib-loaded liposomes are fromabout 40 nm to about 150 nm in diameter.

In various embodiments, the encapsulated carfilzomib is solubilized inthe internal aqueous compartment. In various embodiments, thecarfilzomib is in the form of a suspension. In an exemplary embodiment,the carfilzomib fraction is partially solubilized and partially in theform of a suspension.

As will be appreciated by those of skill in the art, the individualparameters set forth above are freely combinable in any useful format.

Kits

In an exemplary embodiment, the invention provides a kit containing oneor more components of the liposomes or formulations of the invention andinstructions on how to combine and use the components and theformulation resulting from the combination. In various embodiments, thekit includes a sparingly water-soluble agent in one vessel and aliposome preparation in another vessel. An exemplary liposomepreparation includes a distribution of salt on the outside and inside ofthe lipid membrane to establish and/or maintain an ion gradient, such asthat described herein. Also included are instructions for combining thecontents of the vessels to produce a liposome or a formulation thereofof the invention. In various embodiments, the amount of complex andliposome are sufficient to formulate a unit dosage formulation of theagent.

In an exemplary embodiment, one vessel includes a liposome or liposomesolution, which is used to convert at least part of the contents of avessel of a sparingly water-soluble therapeutic agent formulation (e.g.,of an approved therapeutic agent) into a liquid formulation of theliposome encapsulated therapeutic agent at the point of care foradministration to a subject. In an exemplary embodiment, the contents ofthe vessels are sufficient to formulate a unit dosage formulation of thetherapeutic agent.

In the embodiment in which a unit dosage format is formed, the vesselincludes from about 1 mg to about 500 mg of the therapeutic agent, e.g,from about 1 mg to about 200 mg, e.g., from about 5 mg to about 100 mg,e.g., from about 10 mg to about 60 mg.

In an exemplary embodiment, the approved therapeutic agent iscarfilzomib and it is present in the vessel in an amount of from about40 mg to about 80 mg, e.g., from about 50 mg to about 70 mg. In anexemplary embodiment, the carfilzomib is present in about 60 mg.

Methods of Treatment

In one aspect, the invention provides a method of treating aproliferative disorder, e.g., a cancer, in a subject, e.g., a human, themethod comprising administering a composition that comprises apharmaceutical formulation of the invention to a subject in an amounteffective to treat the disorder, thereby treating the proliferativedisorder.

In one embodiment, the pharmaceutical formulation is administered incombination with one or more additional anticancer agent, e.g.,chemotherapeutic agent, e.g., a chemotherapeutic agent or combination ofchemotherapeutic agents described herein, and radiation.

In one embodiment, the cancer is a cancer described herein. For example,the cancer can be a cancer of the bladder (including accelerated andmetastatic bladder cancer), breast (e.g., estrogen receptor positivebreast cancer; estrogen receptor negative breast cancer; HER-2 positivebreast cancer; HER-2 negative breast cancer; progesterone receptorpositive breast cancer; progesterone receptor negative breast cancer;estrogen receptor negative, HER-2 negative and progesterone receptornegative breast cancer (i.e., triple negative breast cancer);inflammatory breast cancer), colon (including colorectal cancer), kidney(e.g., transitional cell carcinoma), liver, lung (including small andnon-small cell lung cancer, lung adenocarcinoma and squamous cellcancer), genitourinary tract, e.g., ovary (including fallopian tube andperitoneal cancers), cervix, prostate, testes, kidney, and ureter,lymphatic system, rectum, larynx, pancreas (including exocrinepancreatic carcinoma), esophagus, stomach, gall bladder, thyroid, skin(including squamous cell carcinoma), brain (including glioblastomamultiforme), head and neck (e.g., occult primary), and soft tissue(e.g., Kaposi's sarcoma (e.g., AIDS related Kaposi's sarcoma),leiomyosarcoma, angiosarcoma, and histiocytoma).

In an exemplary embodiment, the cancer is multiple myeloma or a solidtumor. In one embodiment, the pharmaceutical formulation of theinvention includes carfilzomib as the sparingly water-solubletherapeutic agent.

In one aspect, the disclosure features a method of treating a disease ordisorder associated with inflammation, e.g., an allergic reaction or anautoimmune disease, in a subject, e.g., a human, the method comprises:administering a composition that comprises a pharmaceutical formulationof the invention to a subject in an amount effective to treat thedisorder, to thereby treat the disease or disorder associated withinflammation.

In one embodiment, the disease or disorder associated with inflammationis a disease or disorder described herein. For example, the disease ordisorder associated with inflammation can be for example, multiplesclerosis, rheumatoid arthritis, psoriatic arthritis, degenerative jointdisease, spondouloarthropathies, gouty arthritis, systemic lupuserythematosus, juvenile arthritis, rheumatoid arthritis, osteoarthritis,osteoporosis, diabetes (e.g., insulin dependent diabetes mellitus orjuvenile onset diabetes), menstrual cramps, cystic fibrosis,inflammatory bowel disease, irritable bowel syndrome, Crohn's disease,mucous colitis, ulcerative colitis, gastritis, esophagitis,pancreatitis, peritonitis, Alzheimer's disease, shock, ankylosingspondylitis, gastritis, conjunctivitis, pancreatitis (acute or chronic),multiple organ injury syndrome (e.g., secondary to septicemia ortrauma), myocardial infarction, atherosclerosis, stroke, reperfusioninjury (e.g., due to cardiopulmonary bypass or kidney dialysis), acuteglomerulonephritis, vasculitis, thermal injury (i.e., sunburn),necrotizing enterocolitis, granulocyte transfusion associated syndrome,and/or Sjogren's syndrome. Exemplary inflammatory conditions of the skininclude, for example, eczema, atopic dermatitis, contact dermatitis,urticaria, scleroderma, psoriasis, and dermatosis with acuteinflammatory components. In some embodiments, the autoimmune disease isan organ-tissue autoimmune diseases (e.g., Raynaud's syndrome),scleroderma, myasthenia gravis, transplant rejection, endotoxin shock,sepsis, psoriasis, eczema, dermatitis, multiple sclerosis, autoimmunethyroiditis, uveitis, systemic lupus erythematosis, Addison's disease,autoimmune polyglandular disease (also known as autoimmune polyglandularsyndrome), or Grave's disease.

In another embodiment, a pharmaceutical formulation of the invention ormethod described herein may be used to treat or prevent allergies andrespiratory conditions, including asthma, bronchitis, pulmonaryfibrosis, allergic rhinitis, oxygen toxicity, emphysema, chronicbronchitis, acute respiratory distress syndrome, and any chronicobstructive pulmonary disease (COPD). The pharmaceutical formulation ofthe invention, particle or composition may be used to treat chronichepatitis infection, including hepatitis B and hepatitis C.

In one aspect, the disclosure features a method of treatingcardiovascular disease, e.g., heart disease, in a subject, e.g., ahuman, the method comprising administering a a pharmaceuticalformulation of the invention to a subject in an amount effective totreat the disorder, thereby treating the cardiovascular disease.

In one embodiment, cardiovascular disease is a disease or disorderdescribed herein. For example, the cardiovascular disease may becardiomyopathy or myocarditis; such as idiopathic cardiomyopathy,metabolic cardiomyopathy, alcoholic cardiomyopathy, drug-inducedcardiomyopathy, ischemic cardiomyopathy, and hypertensivecardiomyopathy. Also treatable or preventable using a pharmaceuticalformulation of the inventions, particles, compositions and methodsdescribed herein are atheromatous disorders of the major blood vessels(macrovascular disease) such as the aorta, the coronary arteries, thecarotid arteries, the cerebrovascular arteries, the renal arteries, theiliac arteries, the femoral arteries, and the popliteal arteries. Othervascular diseases that can be treated or prevented include those relatedto platelet aggregation, the retinal arterioles, the glomerulararterioles, the vasa nervorum, cardiac arterioles, and associatedcapillary beds of the eye, the kidney, the heart, and the central andperipheral nervous systems. Yet other disorders that may be treated withpharmaceutical formulation of the invention, include restenosis, e.g.,following coronary intervention, and disorders relating to an abnormallevel of high density and low density cholesterol.

In one embodiment, the pharmaceutical formulation of the invention canbe administered to a subject undergoing or who has undergoneangioplasty. In one embodiment, the Pharmaceutical formulation of theinvention, particle or composition is administered to a subjectundergoing or who has undergone angioplasty with a stent placement. Insome embodiments, the pharmaceutical formulation of the invention,particle or composition can be used as a strut of a stent or a coatingfor a stent.

In one aspect, the invention provides a method of treating a disease ordisorder associated with the kidney, e.g., renal disorders, in asubject, e.g., a human, the method comprises: administering apharmaceutical formulation of the invention to a subject in an amounteffective to treat the disorder, thereby treating the disease ordisorder associated with kidney disease.

In one embodiment, the disease or disorder associated with the kidney isa disease or disorder described herein. For example, the disease ordisorder associated with the kidney can be for example, acute kidneyfailure, acute nephritic syndrome, analgesic nephropathy, atheroembolicrenal disease, chronic kidney failure, chronic nephritis, congenitalnephrotic syndrome, end-stage renal disease, good pasture syndrome,interstitial nephritis, kidney damage, kidney infection, kidney injury,kidney stones, lupus nephritis, membranoproliferative GN I,membranoproliferative GN II, membranous nephropathy, minimal changedisease, necrotizing glomerulonephritis, nephroblastoma,nephrocalcinosis, nephrogenic diabetes insipidus, nephrosis (nephroticsyndrome), polycystic kidney disease, post-streptococcal GN, refluxnephropathy, renal artery embolism, renal artery stenosis, renalpapillary necrosis, renal tubular acidosis type I, renal tubularacidosis type II, renal underperfusion, renal vein thrombosis.

In an exemplary embodiment, the invention provides a method of treatingmetal toxicity or metal overload. Examples of diseases or disordersassociated with metal include iron overload disorders (e.g., thalassemiaor sickle cell anemia), copper over load disorders (e.g., Wilson'sdisease), and radioisotope contamination (e.g., occurring subsequent tocontamination with plutonium, uranium and other radioistopes).

An “effective amount” or “an amount effective” refers to an amount ofthe pharmaceutical formulation of the invention which is effective, uponsingle or multiple dose administrations to a subject, in treating acell, or curing, alleviating, relieving or improving a symptom of adisorder. An effective amount of the composition may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the compound to elicit a desired responsein the individual. An effective amount is also one in which any toxic ordetrimental effects of the composition are outweighed by thetherapeutically beneficial effects.

As used herein, the term “prevent” or “preventing” as used in thecontext of the administration of an agent to a subject, refers tosubjecting the subject to a regimen, e.g., the administration of apharmaceutical formulation of the invention such that the onset of atleast one symptom of the disorder is delayed as compared to what wouldbe seen in the absence of the regimen.

As used herein, the term “subject” is intended to include human andnon-human animals. Exemplary human subjects include a human patienthaving a disorder, e.g., a disorder described herein, or a normalsubject. The term “non-human animals” includes all vertebrates, e.g.,non-mammals (such as chickens, amphibians, reptiles) and mammals, suchas non-human primates, domesticated and/or agriculturally usefulanimals, e.g., sheep, dog, cat, cow, pig, etc.

As used herein, the term “treat” or “treating” a subject having adisorder refers to subjecting the subject to a regimen, e.g., theadministration of a pharmaceutical formulation of the invention suchthat at least one symptom of the disorder is cured, healed, alleviated,relieved, altered, remedied, ameliorated, or improved. Treating includesadministering an amount effective to alleviate, relieve, alter, remedy,ameliorate, improve or affect the disorder or the symptoms of thedisorder. The treatment may inhibit deterioration or worsening of asymptom of a disorder.

The following examples are provided to illustrate exemplary embodimentsof the invention and are not to be construed as limiting the scope ofthe invention.

EXAMPLES Example 1 Carfilzomib Liposome Entrapment by Remote LoadingMaterials and Method

Ammonium sulfate solution was prepared by dissolving ammonium sulfatesolid to a final concentration of 250 mM (500 mequivilents of anion/L)no pH adjustment was made to yield a final pH of 5.6. Sodium sulfatesolution (250 mM) was prepared by adding 0.35 g sodium sulfate to 10 mLdeionized water.

The liposomes were formed by extrusion. Lipids were dissolved in ethanolat a concentration of 500 mM HSPC (591 mg/mL total lipid) at 65° C. andthe 9 volumes of the trapping agent solution heated to 65° C. was addedto the ethanol/lipid solution also at 65° C. The mixture was vortexedand transferred to a 10 mL thermostatically controlled (65° C.) LipexExtruder. The liposomes were formed by extruding 10 times throughpolycarbonate membranes having 0.1 um pores. After extrusion theliposomes were cooled on ice. The transmembrane electrochemical gradientwas formed by purification of the liposomes by dialysis in dialysistubing having a molecular weight cut off of 12,000-14,000. The samplesare dialyzed against 5 mM HEPES, 10% sucrose pH 6.5 (stirring at 4° C.)at volume that is 100 fold greater than the sample volume. The dialysatewas changed after 2 h then 4 more times after 12 h each. Theconductivity of the liposome solution was measured and wasindistinguishable from the dialysis medium ˜40 μS/cm.

The lipid concentration is determined by measuring the cholesterol byHPLC using an Agilent 1100 HPLC with and Agilent Zorbax 5 um, 4.6×150mM, Eclipse XDB-C8 column and a mobile phase of A=0.1% TFA, B=0.1%TFA/MeOH with an isocratic elution of 99% B. The flow rate is 1.0mL/min, column temperature is 50° C., 10 μL injection and detection byabsorbance at 205 nm. The retention time of cholesterol is 4.5 min. Theliposome size is measured by dynamic light scattering.

Carfilzomib (Selleck Chemicals) was dissolved in DMSO at a concentrationof 10 mg/mL. The carfilzomib was introduced to the liposomes at acarfilzomib to HSPC ratio of 100 g drug/mol HSPC (drug to total lipidratio (wt/wt) of 0.12). The liposomes were diluted with 50 mM citrate,10% sucrose pH 4.0 to increase the volume to a point where afteraddition of the drug the final DMSO concentration is 2%. Thecarfilzomib/DMSO was added to the diluted liposomes, which were mixed atroom temperature then transferred to a 65° C. bath and swirled every 30s for the first 3 min and then swirled every 5 min over a total heatingtime of 30 min. All samples were very cloudy when the drug was added andall became clear (same as liposomes with no drug added) after 15 min.After heating for 30 min all samples were placed on ice for 15 min. Theloaded liposomes were vortexed and 100 μL of sample was kept as the“before column” and the rest transferred to microcentrifuge tubes andspun at 10,000 RPM for 5 min. The supernatants were purified on aSephadex G25 column collected and analyzed by HPLC. The HPLC analysis ofcarfilzomib was performed on the same system as described for analysisof cholesterol. The mobile phase consists of A=0.1% TFA, B=0.1% TFA/MeOHwith a gradient elution starting at 50% B and increasing to 83% B in 13min with 7 min equilibration back to 50% B. The flow rate is 1.0 mL/min,column temperature is 30 C, 10 μl injection and detection by absorbanceat 205 nm. The retention time of carfilzomib is 12.2 min. The lipidconcentration is determined by analysis of the cholesterol by HPLC.

Results

The loading of liposomes containing 250 mM ammonium sulfate resulted ina final drug to lipid ratio of 95.26±3.47 g drug/mol of HSPC liposomeswhen the drug was added at 100 g drug/mol of HSPC lipid (95.26±3.47%efficient) and a final drug to lipid ratio of 136.9±7.35 g drug/mol ofHSPC liposomes when the drug was added at 200 g drug/mol of HSPC lipid(67.94±3.67% efficient) (FIG. 2). This demonstrates that the loadingcapacity of these particular liposomes is between 100 and 200 g drug/molphospholipid. The control liposomes containing 250 mM sodium sulfatewhich have no electrochemical gradient for remote loading resulted in afinal drug load of 33.28±0.79 and 29.01±0.79 g drug/mol of HSPC when thedrug was added at a ratio of 100 and 200 g drug/mol of HSPCrespectively. This demonstrates that the capacity for loading theseliposomes was saturated below 100 g drug/mol of HSPC and at this druginput ratio the remote loaded liposomes exhibit >3 fold higher loadingcapacity. Saturation of the drug loading capacity for sodium sulfateliposomes at a ratio at least 3 fold lower than the ammonium sulfateliposomes indicates that when no electrochemical gradient is present forremote loading the drug partitions into the lipid bilayer but does notform a salt with the interior trapping agent. FIG. 5 illustrates theprecipitate is still present after the loading process with sodiumsulfate liposomes but not with ammonium sulfate liposomes.

Conclusion

Liposomes of identical lipid matrix composition and size but varying inthe composition of the sulfate salt internally trapped had verydifferent capabilities to load carfilzomib. The liposome capable ofgenerating an electrochemical gradient (ammonium sulfate) was able toload close to 100% of the drug at optimal conditions and the oneincapable of creating a gradient had poor loading efficiency suggestingthat remote or active loading was the primary mechanism for carfilizomibincorporation into the liposome.

Example 2 Comparison of Liposome Trapping Agents Introduction

Liposomes to be used for remote loading are formed in an ionic solutionthat is intended to complex the loaded drug as a salt. Trapping agentscan form complexes with loaded drugs and the stability of this complexis one factor that dictates liposome drug loading ability, stability anddrug release rates. Comparison of different liposome trapping agents wasmade by evaluating the efficiency of carfilzomib loading.

Methods

Three liposome formulations were used, all at a molar ratio 3 HSPC/2Chol/0.15 PEG-DSPE each with a different trapping agent: 1. melliticacid; 2. ammonium sulfate; and 3. napthelene disulfononic acid.

Mellitic acid (MA) was dissolved in water and titrated with diethylamineto a final pH of 5.5 and concentration of 83 mM (500 mequivilents ofanion/L). Ammonium sulfate was prepared by dissolving ammonium sulfatesolid to a final concentration of 250 mM (500 mequivilents of anion/L)no pH adjustment was made to yield a final pH of 5.6.

Napthelenedisulfonic acid (NDS) was dissolved in water and titrated withdiethylamine to a final pH of 8.0 and concentration of 250 mM (500mequivilents of anion/L).

See Example 1, Carfilzomib liposome entrapment by remote loading fordetails on how the liposomes were made, purified and characterized.

TABLE 1 Sizes of Liposomes Loaded with Carfilzomib. before lyophilizingTrapping agent Z-ave (nm) PDI (NH4)₂SO₄ 108 0.062 (Drug added quickly)(NH4)₂SO₄ 109.2 0.035 (Drug added slowly) Napthelenedisulfonic acid111.9 0.039 Mellitic Acid 105.5 0.08

To ensure complete removal of the DMSO added with carfilzomib, theliposomal carfilzomib samples were dialyzed in dialysis tubing having amolecular weight cut off of 12,000-14,000. The samples are dialyzedagainst 5 mM HEPES, 10% sucrose pH 6.5 (stirring at 4° C.) at volumethat is 100 fold greater than the sample volume. The dialysate waschanged after 2 h then 2 more times after 12 h each. The carfilzomibliposomes were again analyzed for drug and lipid concentration asdescribed above.

Results

The efficiency of carfilzomib remote loading into liposomes at 100 gdrug/mol of HSPC lipid for liposomes with the trapping agents melliticacid, ammonium sulfate, and napthelenedisulfonic acid were 37.4%±2.01%,97.0%±2.38%, and 95.1%±1.76% respectively (FIG. 3).

Conclusion

The invention described here enables remote loading of carfilzomib froman insoluble precipitate into liposomes can be accomplished with varioususing the electrochemical gradient generated by various trapping agentsincluding mellitic acid, ammonium sulfate and napthelene disulfononicacid.

Example 3 Comparison of Method for Introducing Drug Method

A comparison of the method used for addition of the drug to theliposomes during the loading procedure. The loading procedure was thesame as described above in Example 1 with the exception of the drugbeing added to the liposome loading solution as a solid powder, followedby the addition of DMSO to a final concentration of 2% (vol/vol), as a10 mg/mL DMSO solution quickly and as 10 mg/mL DMSO solution slowly(both at a final DMSO content of 2% (vol/vol).

Results

The efficiency of carfilzomib remote loading into liposomes at 100 gdrug/mol of HSPC lipid for the drug which was first added as the solidpowder was 3.88%±0.053% and 3.47%±0.030% when heated to 65° C. for 30and 120 min respectively. The efficiency of loading the drug frominitial stock solutions of 10 mg/mL DMSO solution was 97.0%±2.38% whenthe drug/DMSO was added quickly and 96.3%±1.09% when the drug/DMSO wasadded in 5 increments over 1 min to a liposome solution while vortexing.The drug/liposome mixture that results from the slow drug addition isclearer than the drug/liposome mixture that results from rapid additionof the drug. However, both solutions have no visible precipitate (orcentrifugal precipitate at 10,000 rpm for 5 min) after heating to 65° C.for 30 min, which is a result of all of the drug being loaded into theliposomes regardless of the precipitate formed upon addition of the drug(FIG. 4). This shows that having the drug initially fully dissolved inthe aprotic solvent prior to diluting beyond its solubility limit intoan aqueous solution is useful for remote loading into liposomescontaining a pH or ion gradient.

Example 4

Carfilzomib Loading into Liposomes at Room Temperature

Introduction

The ability to load a drug into liposomes at room temperature isbeneficial to reduce heat-induced drug degradation, simplifymanufacturing and allow for bedside liposome loading. Efficienttransport across the liposome membrane requires the membrane to be in afluid phase. This is accomplished with saturated phospholipids having ahigh phase transition temperature (T_(m)) such as HSPC (T_(m)=55° C.) byheating the liposomes above the T_(m) during the loading process. Analternative to heating is to use lipids that are fluid phase at roomtemperature. The disadvantage of these lipids is that they are unstablein circulation and result in rapid drug release. Sterol modified lipidsincorporate a novel lipid construction where cholesterol (sterol) iscovalently attached to the phosphate headgroup. Sterol modified lipidshave proven to render the sterol non-exchangable from the lipid bilayerin circulation. Sterol modified lipids are also fluid phase at roomtemperature, making them ideal for room temperature loading of drugsinto liposomes that are to be used for in vivo delivery of therapeutics.

Method

The loading of carfilzomib into liposomes at room temperature wasperformed by using two liposome formulations composed of a molar ratioof 95 PChemsPC/5 PEG-DSPE and another with a molar ratio of 3 POPC/2Chol/0.15 PEG-DSPE each containing 250 mM ammonium sulfate as thetrapping agent. The liposomes were prepared using the procedure,drug/liposome ratio, buffers ad pH as described in Example 1. Theliposomes were stirred at room temperature (20° C.) and the carfilzomibwas added as a 10 mg/mL DMSO solution in 5 increments over 1 min toresult in a cloudy solution. The liposome/drug mixture was stirred atroom temperature for a total of 30 min to yield a clear solution withthe same appearance as the liposome solution before the drug was added.

Results

The efficiency of carfilzomib remote loading into liposomes at 100 gdrug/mol of PChemsPC was 95.5%±1.23% The efficiency of carfilzomibremote loading into liposomes at 187 g drug/mol of 3 POPC/2 Chol/0.15PEG-DSPE was 100.52%±1.01%

Conclusion

The invention described here was not able to load carfilzomib intoliposomes by adding the crystal form of the drug directly to the loadingsolution. The drug requires solubilization in some solvent prior toaddition to the loading solution at a concentration above the solubilityof the drug. Liposome loading efficiency of the precipitate that isformed upon addition of the drug to the loading solution is notdependent upon the rate of addition in this case using carfilzomib.

Example 5

Drug Precipitate Loading into Liposomes as Determined by LightScattering at 600 nm.

Introduction

Liposome loading of drug from a precipitate into liposomes is evidencedby the resulting drug to lipid ratio and clarifying of the solution asthe drug precipitate transfers into the liposome. To get a quantitativemeasure liposome loading from a drug precipitate the light scatteringwas measured at 600 nm during the loading process.

Method

Liposomes containing 250 mM ammonium sulfate as trapping agent and 250mM sodium sulfate as control liposomes which would not remote load drug.The liposomes were prepared and loaded using the procedure described inExample 1 except a disposable polystyrene cuvette was used as thereaction vessel. The scattering of light at 600 nm was measured with aUV/vis spectrophotometer during the loading process.

Results/Conclusion

The sodium sulfate liposomes do not show any clarification of theprecipitate during the loading procedure indication that the drug is notremote loading into the liposomes. (see FIG. 5). The ammonium sulfateliposomes efficiently load the drug resulting in clarification of thesolution within 15 min.

Example 6

Confirmation of Drug Release from Remote Loaded Liposomes

Introduction

A reverse gradient was used to attempt to release the active drug fromwithin the liposome. The theory is that if a drug can be released fromwithin a liposome with a reverse gradient there is a high probabilitythe that drug release will occur in vivo.

Method

Liposomes were loaded with carfilzomib as described in Example 1 andwere purified into deionized water. The sample was divided into twoaliquots. To the first aliquot, concentrated Hepes pH 7.4 and NaCl wasadded so that the final concentration was 5 mM Hepes, 145 mM NaCl (HBS).To the second aliquot, concentrated ammonium sulfate was added so thatthe final concentration was 250 mM. No obvious physical changes wereinitially observed. The samples were then heated at 65° C. for 30 min.The samples were transferred to clean eppendorf tubes and centrifugedfor 10,000 rpm for 5 min after which the supernatants and precipitateswere separated and tested for carfilzomib content by HPLC assay.Released drug precipitated, liposome encapsulated drug remained in thesupernatant. The % carfilzomib released was calculated by

${\% \mspace{14mu} {Release}} = \frac{{{amt}.\mspace{14mu} {of}}\mspace{14mu} {drug}\mspace{14mu} {in}\mspace{14mu} {precipitate}}{{{amt}.\mspace{14mu} {of}}\mspace{14mu} {total}\mspace{14mu} {drug}}$

Results

TABLE 2 The reverse gradient-directed drug release from liposomesSolution Composition % Carfilzomib Released Hepes buffered saline 10.6 ±0.28 Ammonium sulfate 68.5 ± 1.82

The drug released using the reverse gradient is 6.5-fold greater thanthe drug released from the control with no reverse gradient (Table 2).HPLC chromatogram of the released drug was identical to the startingmaterial indicating that no degradation of carfilzomib had taken place(FIG. 7). HPLC retention time for the stock solution of carfilzomib was12.2 min and the retention time for the carfilzomib that was releasedfrom the remote loaded liposome was 12.3 min, as shown in FIG. 6. Thesetwo separations times are within the variability of the HPLC system andare not statistically different from each other.

Conclusion

Carfilzomib was released from the liposome using a reverse gradient toyield the original molecule as indicated by HPLC analysis.

Example 7 Carfilzomib Loading as a Function of DMSO Content Introduction

The physical form of the drug when added to liposomes is important forloading efficiency, i.e., when added as a dry powder almost no loadingis observed but addition using a predissolved solution in an aproticsolvent can lead to high entrapment efficiency. This study looks at theeffect of aprotic solvent concentration on drug loading efficiency ofcarfilzomib.

Method

Ammonium sulfate containing liposomes were diluted in 50 mM citric acidsucrose (10% wt/wt) buffer pH 4.0 to 1 mM phospholipid. Various amountsof DMSO were added so that when 200 μg drug was added from a 10mg/mLcarfilzomib solution in DMSO the final DMSO concentration ranged from1-10% v/v.

Results

DMSO had a dramatic effect on the ability of carfilzomib to remote loadinto liposomes. When absent, there is practically no loading. Atconcentrations 1% and above the loading efficiency ranges from 74-94%,with higher efficiencies observed at higher DMSO concentrations (FIG.7). It should be noted that drug precipitates were observed in allsamples before loading commenced, suggesting that the concentrations ofDMSO used here are below the minimum concentration required toeffectively solubilize carfilzomib at the drug concentration used (0.2mg/mL).

Conclusions

The introduction of pre-solubilized carfilzomib is necessary forefficient remote loading. However, above 1% DMSO there is a relativelysmall change in loading efficiency, up as far as 10%.

Example 8 Carfilzomib Solubility as a Function of DMSO ContentIntroduction

Under the conditions described above, carfilzomib is solubilized in DMSObefore diluting in liposome buffer solution prior to loading. It thenimmediately precipitates before remote loading. This study is designedto determine the DMSO concentration that is required to effectivelysolubilize carfilzomib at room temperature and at the temperaturerequired for liposome loading into liposomes composed of high Tm lipids(65° C.).

Method

Carfilzomib was added from a stock 10 mg/mL solution in DMSO to 1 mL ofa citric acid/DMSO mixture so that the composition of DMSO was 2%, 25%,50%, 75% and 100%. The final drug concentration was 0.2 mg/mL. Thesolutions were prepared and measured for optical density at 600 nm. Theoptical density at 600 nm is a good measure of how turbid or how muchscattering material (such as drug precipitates) are in a solution,generally, the more precipitates the higher the absorbance. From FIG. 8is apparent that at DMSO concentrations below 50% vol/vol (25° C.) and25% vol/vol (65° C.) the drug remains in a precipitated form. Only whenthe concentration of DMSO is increased does it become effectivelysolubilized at this concentration of 0.2 mg/mL.

To test the integrity of the liposomes in 25% DMSO we attempted toremote load the water-soluble weak base drugs doxorubicin and17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) andcompared to the same loading without DMSO. We found that the loadingefficiency was adversely affected (Table 3).

Results

At 0.2 mg/mL carfilzomib the drug precipitates and the aggregates arelarge enough to cause a light scattering signal at 600 nm. As the % DMSOis increased the signal is reduced and indicates solubilization of thedrug. We observed that >25% vol/vol DMSO is required to completelydissolve the drug at 0.2 mg/mL at a temperature of 65° C.

TABLE 3 Comparison of remote loading doxorubicin and 17-DMAG intoammonium sulfate containing liposomes in the presence and absence of 25%DMSO. Drug % DMSO % Efficiency compared to control of no DMSOdoxorubicin 25 92.7 ± 0.43 17-DMAG 25 73.1 ± 1.4 

Conclusions

Previous studies loading carfilzomib were done using 10% v/v DMSO orless and the light scattering results above show that the vast majorityof the drug under these conditions is in a precipitated form at theconcentrations used. Adding enough aprotic solvent to completelysolubilize the drug (i.e. greater than 25% DMSO at 65° C.) has anegative impact of the liposome loading of amphipathic weak base drugsindicating liposome instability caused by contents leakage orelectrochemical gradient dissipation for example. Under conditions thatmaintain good liposome stability, we have not found a DMSO concentrationthat will solubilize carfilzomib completely or alternatively we have notfound conditions using DMSO where simultaneously the drug is completelysolubilized and the liposomes are not adversely destabilized.

Example 9 Effect of Delay on Liposome Loading of Carfilzomib After theDrug Precipitate is Formed Introduction

The invention described in this application allows for loading of aninsoluble drug precipitate into liposomes. Example 9 evaluates theeffect of the time between the formation of the drug precipitate and thetime it is loaded into liposomes.

Procedure

Liposomes were prepared from the same composition and methods asdescribed in Example 1.

Carfilzomib was dissolved in DMSO at a concentration of 10 mg/mL and weadded to a final concentration of 2% (v/v) to 50 mM citrate, 10% sucroseat pH 3.5 containing no liposomes. Upon addition of the drug to thecitrate buffer a precipitate was formed. The liposomes for loading wereadded to the solution containing drug precipitate either immediatelyafter formation, after a 1 h delay or after a 12 h delay and then theprecipitate was loaded into the liposomes using the loading conditionsdescribed in Example 1.

Results/Conclusion

The time between the formation of the drug precipitate and the loadingof the precipitate does not have a significant impact on the efficiencyof the liposome loading procedure for carfilzomib even if the delay timeis up to 12 h.

Example 10

Effect of Liposome Drug Payload on Efficiency of Carfilzomib Loaded fromPrecipitate Procedure

Liposomes were prepared from the same composition and methods asdescribed in Comparison of Trapping Agents except the concentration ofammonium sulfate internal trapping agent was either 250 mM or 500 mM.

Carfilzomib was dissolved in DMSO at a concentration of 10 mg/mL. Thecarfilzomib was introduced to the liposomes at carfilzomib to HSPCratios of 91.8, 167, 251, 338 and 433, g drug/mol HSPC for the liposomeshaving 250 mM ammonium sulfate as the trapping agent and 451, 546, 639,and 759 g drug/mol HSPC for the liposomes having 500 mM ammonium sulfateas the trapping agent. The liposomes were diluted with 50 mM citrate,10% sucrose pH 4.0 to increase the volume to a point where afteraddition of the drug the final DMSO concentration is 10%. Thecarfilzomib/DMSO was added to the diluted liposomes, which were mixed atroom temperature then transferred to a 65° C. bath and swirled every 30s for the first 3 min and then swirled every 5 min over a total heatingtime of 30 min. All samples were very cloudy when the drug was added andall became clear (same as liposomes with no drug added) after 15 min.After heating for 30 min all samples were placed on ice for 15 min. Theloaded liposomes were purified and analyzed as described in Example 1.

Results/Conclusion

TABLE 4 Effect of Ammonium Sulfate Trapping Agent Concentration onLiposome Drug Payload of Carfilzomib Loaded from Precipitate Input Drugtrapping Output payload/carrier drug/ agent drug/ weight ratio HSPC SD[(NH₄)₂SO₄] HSPC SD efficiency SD (g drug/g total (g/mol) (g/mol) (mM)(g/mol) (g/mol) % (g/mol) lipid) 91.8 0.3 250 83.5 2.7 90.9 3.0 0.07167.3 4.2 250 127.2 1.5 76.0 2.1 0.11 251.8 5.8 250 174.1 3.9 69.1 2.20.15 338.1 4.1 250 210.7 2.5 62.3 1.1 0.18 432.8 14.4 250 240.5 4.4 55.62.1 0.20 450.6 9.6 500 345.2 7.1 76.6 2.3 0.29 545.9 17.1 500 380.9 21.469.8 4.5 0.32 639.2 42.5 500 438.9 10.2 68.7 4.8 0.37 758.7 12.4 500468.2 4.9 61.7 1.2 0.40

The resulting drug payload increases as the drug to liposome input lipidratios is increased in the loading solution. The efficiency is greatestat the lowest input ratio used for each different concentration ofammonium sulfate trapping agent. Using the conditions described in thisexample, carfilzomib can be loaded into liposomes from an insolubleprecipitate up to a final drug payload of 469±4.9 g drug/mol HSPC(drug/carrier total lipid weight ratio of 0.4) at an efficiency of61.7±1.2%.

Example 11

Loading of Insoluble Carfilzomib into Liposomes Using a TriethylammoniumSulfate Gradient

Introduction

Remote loading of drugs into liposomes is commonly accomplished using anammonium sulfate gradient. Some drug molecules including the examplecarfilzomib used here have an epoxide group which is required foractivity. The epoxide of these drugs is potentially susceptible toaminolysis from any remaining ammonia that is used in the ammoniumsulfate remote loading. In this, Example 11, the ammonium sulfate isreplaced with a triethylammonium sulfate remote loading agent toeliminate potential ammonia/epoxide reactions by replacement withnonreactive triethylamine.

Methods

The liposomes were prepared by using the same compositions and procedureas described in Carfilzomib Liposome Entrapment by Remote Loading withthe following exception that 50 mM triethylammonium sulfate was used asthe trapping agent. Triethylammonium Sulfate was prepared by titrating 1M sulfuric acid with triethylamine to a final pH of 7.3 and sulfateconcentration of 500 mM.

Carfilzomib was dissolved in DMSO at a concentration of 10 mg/mL. Thecarfilzomib was introduced to the liposomes at carfilzomib to HSPCratios of 650 g drug/mol HSPC. The liposomes were diluted with 50 mMcitrate, 10% sucrose pH 4.0 to increase the volume to a point whereafter addition of the drug the final DMSO concentration is 10%. Thecarfilzomib/DMSO was added to the diluted liposomes, which were mixed atroom temperature then transferred to a 65° C. bath and swirled every 30s for the first 3 min and then swirled every 5 min. A sample of theloading mixture was removed at 10, 20, 30 and 40 min during the loadingprocedure and placed on ice for 15 min. The loaded liposomes werevortexed and 100 μL of sample was kept as the “before column” and therest transferred to microcentrifuge tubes and spun at 10,000 RPM for 5min. The supernatants were purified on a Sephadex G25 column collectedand analyzed by HPLC. The drug precipitate pellets were dissolved inDMSO/MeOH (10:1) and analyzed by HPLC.

Results/Conclusion

Loading an insoluble carfilzomib precipitate into liposomes using atriethylammonium sulfate gradient results in similar liposomes to thoseproduced using an ammonium sulfate gradient (Example 1). FIG. 12illustrates the time dependence on the liposome loading, which beginsquickly by 10 min. The greatest payload achieved was 440±12.6 g drug/molHSPC (efficiency of 65.9±1.98%) was achieved at 30 min. This resultusing 500 mM triethylamine as a trapping agent at drug to HSPC ratios of650 g drug/mol HSPC is very similar to that using 500 mM ammoniumsulfate as the trapping agent drug to HSPC ratios of 639 g drug/mol HSPCwhich resulted in a final drug to lipid ratio of 440±10.2 g drug/molHSPC (efficiency of 68.7±4.80%).

The insoluble drug precipitate on the liposome exterior is transferred(remote loaded) to the liposome interior as indicated a reduction in theamount of precipitate in the mixture over the course of the loadingprocess. FIG. 12 shows the greatest reduction in the extraliposomalprecipitate happens between 0-10 min which corresponds to the loading ofprecipitate into liposomes as seen in FIG. 13.

Example 12

Loading Another Sparingly Soluble Drug from a Precipitate

Introduction

Another drug, aripiprazole is formulated with sulfobutyl cyclodextran(SBCD) and is used to treat bipolar disorders and schizophrenia(Abilify, Pfizer). The drug is very insoluble in water and when added toa liposome suspension, fine precipitates are immediately observed.

Whether aripiprazole would remote load under similar conditions outlinedabove for carfilzomib was tested.

Method

Liposomes (HSPC/Chol/PEG-DSPE 3/2/0.15 mol/mol/mol) containing 250 mMammonium sulfate or 250 mM sodium sulfate were diluted in 1 mL of 50 mMcitric acid, 10% (wt/wt) sucrose, pH 4.0 to a concentration of 6 mMphospholipid. 0.3 mg of aripiprazole was added from a stock solution of15 mg/mL in DMSO, so that the final DMSO concentration was 2% (v/v).Fine precipitates were immediately observed after the drug was added toboth liposome samples. The samples were heated at 65° C. for 30 min, thecooled on ice. The samples were filtered through a 0.2 umpolyethersulfone syringe filter to remove any drug precipitates,followed by purification on a Sephadex G25 column equilibrated with HBS,pH 6.5 to remove any soluble extraliposome drug. The turbid fraction wascollected and analyzed for lipid and drug as described above.

Results

TABLE 5 Results of loading aripiprazole into liposomes containingammonium and sodium sulfate. Loading Input D/L Output D/L Fold IncreaseAgent ug/umol ug/umol % Efficiency NH₄SO₄/NaSO₄ (NH₄)₂SO₄ 50 42.28 ±0.49 84.56 ± 0.99 49.3 (Na)₂SO₄ 50  0.86 ± 0.51  1.71 ± 0.10

The liposomes containing ammonium sulfate were found to loadapproximately 85% of the drug, while the loading into sodium sulfateliposomes was less than 2%, with about a 50-fold increase in loadingattributable to the ability of ammonium sulfate liposomes to facilitateremote loading (Table 5).

Ariprazole, when introduced to the liposome solution in the form of aSBCD complex (from the pharmaceutical product Abilify) gave a loadingefficiency of 68% under the same concentration and loading conditions(FIG. 14).

Conclusion

This is another example of a poorly soluble drug, that can be remoteloaded into liposomes using the approach described above, and givesslightly better loading than if the drug was introduced as a SBDCcomplex.

Example 13

Loading Sparingly Soluble Drug from Precipitates Made by DilutingVarious Drug Solvent solutions into Liposome Solution

This Example describes a technique for remote loading poorly solubledrugs into liposomes that begins with dissolving the drug in asolubilizing agent that initially forms drug precipitates when added toan aqueous solution of liposomes. After some incubation time the drugenters the liposome in response to an electrochemical gradient,accumulating in the liposome core. Solvents that may be used include butnot limited to dimethylsulfoxide, dioxane, tetrahydrofuran,dimethylformamide, acetonitrile, dimethylacetamide, sulfolane, gammabutyrolactone, pyrrolidones, 1-methyl-2-pyrrolidinone, methylpyrroline,ethylene glycol monomethyl ether, diethylene glycol monomethyl ether,polyethylene glycol.

Method

Aripiprazole was dissolved in a range of solvents indicated below at 4mg/mL. Liposomes composed of HSPC/Chol/PEG-DSPE (3/2/0.15 mol/mol/mol)that were prepared in 250 mM ammonium sulfate were used and diluted to 6mM in Hepes buffered sucrose 10% (wt/wt) (HBSuc pH 6.5). 0.3 mg of drugwas introduced by slow addition of each solvent while vortexing. Thefinal solvent concentration was 7.5% for all samples. As controls, thedrug was added from each solvent to the same volume of HBSuc pH 6.5without the liposomes. The samples were heated at 65° C. for 30 min thencooled on ice. After reaching room temperature again, the samples weremeasured for absorbance at 600 nm (Cary 100 Bio UV-Vis spectrometer) andthe values are displayed below (FIG. 15).

Results

All the solutions without liposomes became extremely turbid or there wasgross precipitation and settling (especially in the case of methanol and1-butanol). Some of the liposome samples were also very turbid, but someclarified the drug precipitate consistent with earlier resultsindicating drug loading of the drug precipitate had taken place (namelyin the cases where the drug was initially dissolved in DMSO,1-4-methylpyrrolidone, diethylenemonoethylether or polyethyleneglycol(MW400), see FIG. 15.

Example 14 Remote Loading of an Insoluble Precipitate of DeferasiroxInto Liposomes Using an Acetate Gradient

Remote loading of deferasirox into liposomes containing calcium acetatedemonstrates the use of an acetate gradient for loading an ironchelating agent. Calcium acetate gradient remote loading differs fromammonium sulfate remote loading in that the drug molecule being loadedmust have a carboxylate (or hydroxamate) rather than an amine.Deferasirox is known to have significant kidney toxicity and liposomedelivery is a technique for reducing kidney toxicity.

Method

The remote loading of a deferasirox insoluble precipitate into iposomesusing an acetate gradient is performed in the same manner as acetateloading of soluble carboxyfluoroscein and nalidixic acid by Clerc andBarenholtz 1995 (PMID 8541297). Liposomes are prepared as described inExample 1 but in this case the liposomes are extruded in a solution of120 mM calcium acetate at pH 8. The acetate gradient is formed byexchanging the external media for 120 mM sodium sulfate at pH 6.0.Deferasirox is dissolved in DMSO at a concentration of 10 mg/ml andadded to the liposome suspension where it forms a precipitate. Theprecipitate is loaded into the liposomes by heating to 65° C. for 1 hand purification and analysis is performed as described in Example 1.

Results

Deferasirox forms a precipitate when diluted from a 10 mg/ml DMSO stockto a concentration of 1 mg/ml in the liposome loading suspension due toits poor water solubility (˜0.038 mg/mL). The insoluble deferasiroxprecipitate is loaded into the liposomes using a calcium acetategradient at an efficiency at least 5-fold greater than it is loaded intocontrol liposomes which contain sodium sulfate and no acetate gradient.

Remote loading an insoluble precipitate of deferasirox into the liposomeprovides an example of the use of an acetate gradient to remote load acarboxylate drug from a precipitate. In this example the drug beingloaded is a chelating agent, in particular an iron chelating agent. The5-fold greater loading into the liposomes having an acetate gradientover control liposomes indicates that the majority of the deferasirox isremote loaded rather than intercalated in the lipid bilayer.

Example 15 Introduction

One goal of liposomal delivery of carfilzomib is to protect the drugfrom degradation and elimination which required the drug to be retainedwithin the liposome. One technique for evaluating the drug retentionwithin the liposome, and thus the benefits obtained from liposomedelivery, is to measure the pharmacokinetics of the drug in mice. Stableformulations with greater drug retention within the liposome will resultin a higher concentration of non-metabolized drug in mouse plasmacompared to less stable formulations or unencapsulated drug.

Materials and Methods

100 nm liposomes comprised of HSPC/Cholesterol/PEG-DSPE (60/40/5mol/mol/mol) and sphingomyelin/cholesterol/PEG-DSPE (55/45/2.8,mol/mol/mol) were formed, purified and drug loaded with carfilzomibusing the methods described in Example 1. The trapping agents used toremote load carfilzomib were triethylammonium dextran sulfate (1.0 MSO₄) or triethylammonium sucroseoctasulfate (1.0 M SO₄). The drug loadedliposomes were purified by tangential flow filtration with bufferexchange into HBS, pH 6.5. The liposomes were sterile filtered through0.2 um polyethersulfone filters and assayed for carfilzomib and lipidcontent as described in Example 1. The drug-to-lipid ratio, drugconcentration and loading efficiency were calculated and results shownin Table 6.

Results.

TABLE 6 Carfilzomib concentration in mouse plasma after IV.administration of liposome formulations. Lipid Formulation Drug LoadingCFZ/PL # (mol/mol/mol) Trapping Agent Efficiency (μg/μmol) % ID @ 4 h 1HSPC/Chol/PEG- Ammonium Sucrose 94.1 ± 0.43 329.2 ± 2.26 0.65 ± 0.29DSPE (60/40/5) Octasulfate (1.0M SO₄) 2 HSPC/Chol/PEG- Triethylammonium94.6 ± 7.55  381 ± 12.1 4.48 ± 1.10 DSPE (60/40/5) Dextran Sulfate (1.0MSO₄) 3 HSPC/Chol/PEG- Triethylammonium 94.6 ± 7.55  381 ± 12.1  5.53 ±1.69† DSPE (60/40/5) Dextran Sulfate (1.0M SO₄) 4 SM/Chol/PEG-Triethylammonium 80.4 ± 0.71 321.8 ± 7.98 66.3 ± 20.3 DSPE (55/45/2.8)Dextran Sulfate (1.0M SO₄) †formulation #3 is the same as #2 except itwas stored at 4° C. for 30 days before PK analysis

In addition, we examined the pharmacokinetics of carfilzomibencapsulated in the liposome formulations in male CD1 mice. The micewere dosed by IV bolus injection through the tail vein at 5 mg/kgcarfilzomib using 3 mice per formulation. At 4 h, the mice weresacrificed and plasma harvested by centrifugation of the blood. 0.1 mLof plasma was mixed with 0.2 mL methanol, mixed well and carfilzomibconcentration measured by HPLC as described in Example 1. The loadingefficiency, drug/lipid ratio and percent of the injected dose remainingin the plasma 4 hours after a tail vein injection of the liposomecarfilzomib (% ID@ 4 h) are shown in Table 6. While no effort was madeto distinguish between non-liposome entrapped and liposome entrappeddrug in the plasma as our analysis measures total drug content wepresume that the majority of the measured carfilzomib is liposomeentrapped because the drug is very rapidly eliminated in the bloodstream (t_(1/2)<20 min) (Yang et al 2011, Drug Metab Dispos. 2011October; 39(10):1873-82). We observed a 100-fold range of drug retentionfrom 0.65% to 66.3% ID depending on the liposome formulationcomposition. The most stable liposome tested was sphingomyelin based andcontained an internal ammonium dextran sulfate solution. The liposomesdescribed above increased the plasma retention of carfilzomib 46-to-4735fold more than a SBCD formulation, or 5-to-510 fold higher thanpublished liposome formulations at 4 h post administration. (Chu et al2012 AAPS Meeting, Poster T2082).

Example 16 Remote Loading of an Insoluble Precipitate of DeferasiroxInto Liposomes Using an Acetate Gradient Introduction

Remote loading of deferasirox (DFX) into liposomes containing calciumacetate demonstrates the use of an acetate gradient for loading an ironchelating agent. Calcium acetate gradient remote loading differs fromammonium sulfate remote loading in that the drug molecule being loadedmust have a carboxylate (or hydroxamate) rather than an amine.Deferasirox is known to have significant kidney toxicity and liposomedelivery is a technique for reducing kidney toxicity.

Method

Liposomes were prepared using the extrusion and purification methoddescribed in Example 1. The lipid composition was HSPC/Cholesterol(3/0.5, mol/mol) or POPC/cholesterol (3/0.5, mol/mol). The trappingagent consisted of calcium acetate or sodium sulfate each at aconcentration of 120 mM. A solution of DFX in DMSO at 20 mg/mL was addedto the liposome solution slowly over 30 seconds while vortexing toproduce a drug precipitate in the liposome solution. The target drug tophospholipid ratio was 100 g DFX/mol phospholipid. The solution washeated for 30 min (at 45° C. for POPC liposomes and 65° C. for HSPCliposomes) and then cooled on ice. A sample was removed to determine theinput drug to lipid ratio and the remaining solution was spun in acentrifuge at 12,000 RPM for 5 minutes to pellet any unloaded drug. Thesupernatant was further purified from unloaded drug using a Sephadex G25size exclusion column eluted with 5 mM HEPES, 145 mM NaCl at pH 6.5. Thepurified liposomes are analyzed for drug and lipid content by HPLC usingthe system described in Example 1 and a program consisting of gradientelution of 65% B to 98% B in 6 min with 5 min equilibration back to 65%B (A=0.1% TFA, B=0.1% TFA/MeOH, 1.0 mL/min), column temperature heldconstant at 30° C., 10 ul injection, and detection by absorbance at 254nm.

Results

Upon addition of the drug in DMSO to the liposomes containing calciumacetate as the trapping agent, the solution of POPC liposomes were lesscloudy than the solution of HSPC liposomes, both contained precipitateddrug before loading. After heating, the solutions clarified and appearedlike liposomes with no drug precipitate. Liposomes containing sodiumsulfate as the control trapping agent never clarified during the heatingprocess and a drug precipitate pellet was formed upon centrifugation.The loading of liposomes containing calcium acetate made from POPC andHSPC was very efficient. Both liposomes containing the calcium acetatetrapping resulted in >90% loading efficiency. The liposomes containingsodium sulfate resulted in 3.3% loading efficiency, which indicates thatthe loading of DFX into calcium acetate liposomes is not passive but canbe described as remote loading. The DFX loading results are shown inTable 7 (FIG. 16).

TABLE 7 Loading Efficiency of DFX in Calcium Acetate Liposomes 2 DFXloading Lipid composition Interior buffer efficiency 3 mol POPC/0.5 molChol 120 mM calcium acetate 94.8 ± 1.46% 3 mol HSPC/0.5 mol Chol 120 mMcalcium acetate 92.5 ± 0.33% 3 mol HSPC/0.5 mol Chol 120 mM sodiumsulfate  3.3 ± 0.14%

Conclusion

Remote loading an insoluble precipitate of deferasirox into the liposomeprovides an example of the use of an acetate gradient to remote load acarboxylate drug from a precipitate. In this example the drug loaded wasa chelating agent, in particular an iron chelating agent. The 28-foldgreater loading into the liposomes having an acetate gradient overcontrol liposomes indicates that the majority of the deferasirox isremote loaded rather than intercalated in the lipid bilayer.

Example 17

Remote Loading of an Insoluble Precipitate of Deferasirox IntoLiposomes. Evaluation of Drug to Lipid Ratio and Calcium AcetateTrapping Agent Concentration.

Introduction

The remote loading capacity of DFX in liposomes containing calciumacetate was evaluated by using different concentrations calcium acetateon the liposome interior and loading a range of different DFX-to-lipidratios.

Method

Liposomes were prepared using the extrusion and purification methoddescribed in Example 1. The lipid composition was POPC/cholesterol(3/0.5, mol/mol). The trapping agent consisted of calcium acetate 120mM, 250 mM or 500 mM. A solution of DFX in DMSO at 20 mg/mL was added tothe liposome solution slowly over 30 seconds while vortexing to producea drug precipitate in the liposome solution. The target drug tophospholipid ratio was 100, 200 or 300 g DFX/mol phospholipid. Thesolution was heated for 30 min at 45° C. and then cooled on ice. Asample was removed to determine the input drug to lipid ratio and theremaining solution was spun in a centrifuge at 12,000 RPM for 5 minutesto pellet any unloaded drug. The supernatant was further purified fromunloaded drug using a Sephadex G25 size exclusion column eluted with 5mM HEPES, 145 mM NaCl at pH 6.5. The purified liposomes are analyzed fordrug and lipid content by HPLC as described in Example 16.

Results

Upon addition of the drug in DMSO to the liposomes containing calciumacetate as the trapping agent the DFX forms a precipitate beforeloading. After heating, the solutions clarify and look like liposomeswith no drug precipitate. The maximum drug load was higher for liposomescontaining 250 and 500 mM calcium acetate compared to 120 mM calciumacetate. The maximum drug load and efficiency was achieved at an inputof 200 g DFX/mol phospholipid for liposomes containing either 250 mMcalcium acetate or 500 mM calcium acetate. The efficiency of loading fora target of 100 g DFX/mol phospholipid ranged from 99.2 to 103% for allthree concentrations of internal calcium acetate. When the target drugload was increased to 200 g DFX/mol phospholipid the efficiency ofliposomes having 250 or 500 mM internal calcium acetate was at leasttwo-fold greater than liposomes having an internal calcium acetateconcentration of 120 mM. The capacity of all three liposomes wasexceeded at input of 300 g DFX/mol phospholipid resulting in nefficiency <24%. The results are shown in FIG. 17.

Conclusion

The drug payload capacity of DFX when remote loaded into liposomes canbe substantially increased by increasing the concentration of thetrapping agent concentration inside the liposome. This exampledemonstrates the dependence of loading capacity on calcium acetatetrapping agent concentration. This example also demonstrates DFXliposome loading can have an optimum drug to lipid ratio where theefficiency and drug load are both greatest. The achieved drug to lipidratio allows for the DFX to be administered to an animal using atolerated dose of lipid.

Example 18

Remote Loading of an Insoluble Precipitate of Deferasirox IntoLiposomes. Evaluation of Trapping Agent.

The remote loading capacity of DFX in liposomes containing calciumacetate, magnesium acetate and zinc acetate was evaluated by preparingliposomes with different trapping agents on the interior and loading arange of different DFX-to-lipid ratios.

Method

Liposomes were prepared using the extrusion and purification methoddescribed in Example 1. The lipid composition was POPC/cholesterol(3/0.5, mol/mol). The trapping agent consisted of calcium acetate,magnesium acetate or zinc acetate at 120 mM. A solution of DFX in DMSOat 20 mg/mL was added to the liposome solution slowly over 30 secondswhile vortexing to produce a drug precipitate in the liposome solution.The target drug to phospholipid ratio was 100, 150 or 200 g DFX/molphospholipid. The solution was heated for 30 min at 45° C. and thencooled on ice. A sample was removed to determine the input drug to lipidratio and the remaining solution was spun in a centrifuge at 12,000 RPMfor 5 minutes to pellet any unloaded drug. The supernatant was furtherpurified from unloaded drug using a Sephadex G25 size exclusion columneluted with 5 mM HEPES, 145 mM NaCl at pH 6.5. The purified liposomesare analyzed for drug and lipid content by HPLC as described in Example16.

Results

Upon addition of the drug in DMSO to the liposomes, the DFX forms aprecipitate before loading. After heating, the solutions containingliposomes with calcium acetate and magnesium acetate became much lessturbid than the liposomes containing zinc acetate as the trapping agent.The maximum drug load was highest for the liposomes containing magnesiumthe second highest for the liposomes containing calcium acetate and theliposomes containing zinc acetate resulted in the lowest drug payload.The efficiency of loading for a target of 100 g DFX/mol phospholipid was5.3±0.07% using zinc acetate whereas the efficiency using calciumacetate and or magnesium acetate were 97.6±0.41% and 99.2±2.42%,respectively. The results are shown in FIG. 18.

Conclusion

The drug payload capacity of DFX when remote loaded into liposomes canbe dependent on the particular metal salt of acetate used for remoteloading. This example demonstrates that magnesium acetate is a bettertrapping agent for DFX than calcium acetate or zinc acetate, and bothare far superior trapping agents than zinc acetate.

Example 19 Introduction

The loading agent and liposome composition influences how effectivelyliposome formulations containing carfilzomib can be prepared and theliposomes' in vivo pharmacokinetic properties.

Materials and Methods

100 nm liposomes comprised of HSPC/Cholesterol/PEG-DSPE (60/40/5mol/mol/mol) and sphingomyelin/cholesterol/PEG-DSPE (55/45/2.8,mol/mol/mol) were formed, purified and drug loaded with carfilzomibusing the methods described in Example 1. The trapping agents used toremote load carfilzomib were 0.65 M citric acid, 0.65 triethylammoniumcitrate, 0.33 M triethylammonium mellitic acetate, and triethylammoniumnapthalene disulfate (1.0 M SO₄). The drug loaded liposomes werepurified by dialysis with buffer exchange into HBS, pH 6.5. Theliposomes were sterile filtered through 0.2 um polyethersulfone filtersand assayed for carfilzomib and lipid content as described in Example 1.The drug-to-lipid ratio, loading efficiency and percent injected doseremaining in plasma 4 h after a tail injection into a mouse (% ID @ 4 h)were calculated and results shown in Table 8.

Results.

TABLE 8 Carfilzomib liposome loading results and concentration in mouseplasma after IV administration of liposome formulations. Drug LoadingLipid Formulation Efficiency CFZ/PL # (mol/mol/mol) Trapping Agent (%)(μg/μmol) % ID @ 4 h 1 HSPC/Chol/PEG- 0.65M Citric acid Sample N.D. N.D.DSPE (60/40/5) aggregates 2 HSPC/Chol/PEG- 0.65M Sample N.D. N.D. DSPE(60/40/5) triethylammonium aggregates citrate 3 HSPC/Chol/PEG- 0.33MSample N.D. N.D. DSPE (60/40/5) triethylammonium aggregates melliticacetate 4 HSPC/Chol/PEG- Naphthalene disulfate 85.7 ± 4.67 300 ± 16.3non DSPE (60/40/5) (1.0M SO₄) detectable N.D. = not done because thesamples aggregated

Upon addition of the drug in DMSO to the liposomes, the carfilzomibforms a precipitate before loading. After heating, formulations 1-3 inTable 8 showed an increase in turbidity due to large aggregates in theremote loading mixture that did not allow for purification of theliposomes. Formulation 4 of Table 8 became less turbid after heating andresulted in a loading efficiency of 85.7±4.67%. Formulation 4 of Table 8was evaluated for drug retention in plasma four hours after IV injectionin mice. At four hours post injection, there was no detectablecarfilzomib present in the plasma. This is to be contrasted to theresults in table 6 where all four formulations had measureable andsignificant quantities of carfilzomib in plasma at four hourspost-injection.

Conclusion

From inspection of the data in Table 6 and 8 it is clear thatoptimization of liposome formulation for retention of carfilzomib inplasma after intravenous injection in mice requires: precise control ofthe liposome composition and the encapsulation of the appropriate remoteloading agent. Formulations based upon the widely used FDA approvedDoxil® product were inferior to the optimal lipid composition ofSM/Chol/PEG-DSPE (55/45/2.8). The amine salt of dextran sulfate wasencapsulated.

The foregoing descriptions of specific embodiments of the presentinvention have been presented for purposes of illustration anddescription. They are not intended to be exhaustive or to limit theinvention to the precise forms disclosed, and obviously manymodifications and variations are possible in light of the aboveteaching. The embodiments were chosen and described in order to bestexplain the principles of the invention and its practical application,to thereby enable others skilled in the art to best utilize theinvention and various embodiments with various modifications as aresuited to the particular use contemplated. It is intended that the scopeof the invention be defined by the claims appended hereto and theirequivalents.

All publications, patents, and patent applications cited herein arehereby incorporated by reference in their entirety for all purposes.

1-20. (canceled)
 21. A method of remotely loading a solid sparinglywater-soluble active agent into liposomes from an aqueous suspensioncomprising the solid sparingly soluble active agent, said methodcomprising: (a) preparing an aqueous suspension of the liposomes havinga proton or an ion gradient across the liposome membrane; (b) preparinga solution of the sparingly water-soluble agent in a solvent selectedfrom an aprotic solvent and a polyol; (c) forming a final suspension bycontacting the liposome suspension in (a) with the solution in (b), suchthat the sparingly water-soluble active agent precipitates, forming thefinal suspension comprising the solid sparingly water-soluble agent andthe liposomes; and (d) incubating the final suspension of (c) for aperiod of time sufficient for the solid sparingly water-soluble agent tobe remotely loaded into the liposomes.
 22. The method according to claim21, wherein at least about 70% of the solid sparingly water-solubleactive agent is remotely loaded into the liposome.
 23. The methodaccording to claim 21, wherein the liposomes have a size of from about30 nanometers to about 40 microns.
 24. The method according to claim 21,wherein the lipsomes comprise from about 50 mol % to about 70 mol %HSPC, from about 25 mole % to about 50 mole % cholesterol and from about0 mol % to about 10 mol % PEG-DSPE.
 25. The method according to claim24, wherein the liposomes comprise about 60 mol % HSPC, about 40 mol %cholesterol and about 5 mol % PEG-DSPE.
 26. The method according toclaim 21, wherein the liposomes comprise from about 45 mole % to about75 mole % sphingomyelin, from about 25 mole % to about 50 mole %cholesterol and from about 0 mole % to about 10 mole % PEG-DSPE.
 27. Themethod according to claim 26, wherein the liposomes comprise about 55mole % sphingomyelin, about 45 mole % cholesterol and about 2.8 mole %PEG-DSPE.
 28. The method according to claim 21, wherein the sparinglywater-soluble active agent has an aqueous solubility of less than 2mg/mL.
 29. The method according to claim 26, wherein the sparinglywater-soluble active agent has an aqueous solubility at pH 7 of lessthan 2 mg/mL.
 30. The method according to claim 21, wherein the gradientis a gradient of a member selected from an amine or a metal salt of amember selected from a carboxylate, a sulfate, a sulfonate, a phosphatea phosphonate and an acetate.
 31. The method according to claim 28,wherein the gradient is a gradient of a member selected from ammoniumsulfate, triethylammonium sulfate, calcium acetate, magnesium acetate,and ammonium chloride.
 32. The method according to claim 21, wherein thegradient is a gradient of triethylammonium dextran sulfate or ammoniumdextran sulfate.
 33. The method according to claim 21, wherein theloading efficiency (grams encapsulated agent/moles of phospholipid) isat least 90% of the active agent precipitate.
 34. The method accordingto claim 21, wherein the sparingly water-soluble active agent iscarfilzomib, and about 60 mg of the carfilzomib is loaded into fromabout 90 mg to about 200 mg of lipid.
 35. The method according to claim32, wherein about 60 mg of carfilzomib is loaded into about 170 mg oflipid.
 36. The method according to claim 32, wherein the liposomes arefrom about 40 nm to about 150 nm in diameter.
 37. The method accordingto claim 21, further comprising, following (c), isolating said liposomesand suspending them in a pharmaceutically acceptable carrier.
 38. Amethod of remotely loading a solid active agent into liposomes from anaqueous suspension comprising the solid active agent and the liposomes,said agent having a water solubility of less than 2 mg/mL, said methodcomprising: (a) incubating the aqueous suspension of the solid activeagent and the liposomes, said liposomes having a liposome membraneselected from: (i) HSPC/cholesterol/PEG-DSPE (60/40/5); and (ii)sphingomyelin/cholesterol/PEG-DSPE (55/45/2.9),  said aqueous suspensionhaving a proton or an ion gradient across the liposome membrane, for aperiod of time sufficient for the solid active agent to be remotelyloaded into the liposomes.
 39. A method of remotely loading a solidactive agent into liposomes from an aqueous suspension comprising thesolid active agent and the liposomes, said agent having a watersolubility of less than 2 mg/mL, said method comprising: (a) incubatingthe aqueous suspension of the solid active agent and the liposomes, saidliposomes having a liposome membrane selected from: (i)HSPC/cholesterol/PEG-DSPE (60/40/5); and (ii)sphingomyelin/cholesterol/PEG-DSPE (55/45/2.9),  said aqueous suspensionhaving an ammonium ion gradient across the liposome membrane, for aperiod of time sufficient for the solid active agent to be remotelyloaded into the liposomes.
 40. The method according to claim 39, whereinthe sparingly water-soluble active agent is selected from aripiprazole,and deferasirox.